Clock Proteins KaiA Research Articles

Diurnal Regulation of In Vivo Localization and CO2-Fixing Activity of Carboxysomes in Synechococcus elongatus PCC 7942.

Carboxysomes are the specific CO2-fixing microcompartments in all cyanobacteria. Although it is known that the organization and subcellular localization of carboxysomes are dependent on external light conditions and are highly relevant to their functions, how carboxysome organization and function are actively orchestrated in natural diurnal cycles has remained elusive. Here, we explore the dynamic regulation of carboxysome positioning and carbon fixation in the model cyanobacterium Synechococcus elongatus PCC 7942 in response to diurnal light-dark cycles, using live-cell confocal imaging and Rubisco assays. We found that carboxysomes are prone to locate close to the central line along the short axis of the cell and exhibit a greater preference of polar distribution in the dark phase, coupled with a reduction in carbon fixation. Moreover, we show that deleting the gene encoding the circadian clock protein KaiA could lead to an increase in carboxysome numbers per cell and reduced portions of pole-located carboxysomes. Our study provides insight into the diurnal regulation of carbon fixation in cyanobacteria and the general cellular strategies of cyanobacteria living in natural habitat for environmental acclimation.

Mutation of alanine-422 in KaiC leads to a low amplitude of rhythm in the reconstituted cyanobacterial circadian clock.

The cyanobacterial circadian oscillator can be reconstituted by mixing the purified clock proteins KaiA, KaiB, and KaiC with ATP in vitro, leading to a 24-h oscillation of KaiC phosphorylation. The cyanobacterial mutant pr1 carrying valine instead of alanine at position 422 of KaiC (KaiC-A422V) lost the ability to shift the phase of the circadian rhythm. In this study, we analyzed KaiC-A422V to investigate the effect of this single-residue substitution on the in vitro reconstitution of KaiC oscillation. KaiC-A422V exhibited low amplitude oscillations of phosphorylation with a smaller amount of Kai complex than wild-type KaiC (KaiC-WT). Although KaiA can stimulate KaiC phosphorylation, the phosphorylation level of KaiC-A422V is much lower than that of KaiC-WT even at higher KaiA concentrations. It has been suggested that monomer shuffling of KaiC is involved in entraining the in vitro rhythm. To examine whether KaiC-A422V has the capacity for monomer shuffling, we used the difference in the amplitude of the phosphorylation rhythms between KaiC-WT and KaiC-A422V as the indicator of monomer shuffling. When KaiC-A422V and KaiC-WT were mixed, the amplitude of the phosphorylation rhythm changed according to the mixing ratio. This suggests that KaiC-A422V has a reduced ability to shuffle monomers in hexameric KaiC. In addition, the A422V mutation resulted in a change of the stability of the KaiC protein.

Molecular Dynamics Simulations of the Circadian Clock Protein Kaic Reveal Structural Insights into the Nucleotide Release and Circadian Timing Mechanisms

The cyanobacterium S. elongatus keeps track of time partly through three soluble clock proteins KaiA, KaiB, and KaiC. These proteins form a powerful system to study the biophysical basis of circadian rhythms, because an in vitro mixture of the three proteins is sufficient to generate a robust ∼24-hour rhythm in the phosphorylation of KaiC. The nucleotide bound states of KaiC is important in controlling both KaiB binding to the N-terminal domain (CI) and the phosphotransfer reactions that (de)phosphorylate the KaiC C-terminal domain (CII). However, the nucleotide exchange pathways associated with transitions among these states are poorly understood. In this study, we integrate recent advances in molecular dynamics methods to elucidate the pathway for Mg⋅ADP release from the CII domain. We find that nucleotide release is coupled to large-scale conformational changes in the KaiC hexamer. In particular, releasing the nucleotide requires solvating the active site, which in turn requires widening the subunit interface leading to the active site. This conformational rearrangement is linked to the extension of the A-loop, a structure implicated in KaiA binding. These results provide a molecular hypothesis for how KaiA acts as a nucleotide exchange factor.

Molecular dynamics simulations of nucleotide release from the circadian clock protein KaiC reveal atomic-resolution functional insights

The cyanobacterial clock proteins KaiA, KaiB, and KaiC form a powerful system to study the biophysical basis of circadian rhythms, because an in vitro mixture of the three proteins is sufficient to generate a robust ∼24-h rhythm in the phosphorylation of KaiC. The nucleotide-bound states of KaiC critically affect both KaiB binding to the N-terminal domain (CI) and the phosphotransfer reactions that (de)phosphorylate the KaiC C-terminal domain (CII). However, the nucleotide exchange pathways associated with transitions among these states are poorly understood. In this study, we integrate recent advances in molecular dynamics methods to elucidate the structure and energetics of the pathway for Mg·ADP release from the CII domain. We find that nucleotide release is coupled to large-scale conformational changes in the KaiC hexamer. Solvating the nucleotide requires widening the subunit interface leading to the active site, which is linked to extension of the A-loop, a structure implicated in KaiA binding. These results provide a molecular hypothesis for how KaiA acts as a nucleotide exchange factor. In turn, structural parallels between the CI and CII domains suggest a mechanism for allosteric coupling between the domains. We relate our results to structures observed for other hexameric ATPases, which perform diverse functions.

Revealing circadian mechanisms of integration and resilience by visualizing clock proteins working in real time

The circadian clock proteins KaiA, KaiB, and KaiC reconstitute a remarkable circa-24 h oscillation of KaiC phosphorylation that persists for many days in vitro. Here we use high-speed atomic force microscopy (HS-AFM) to visualize in real time and quantify the dynamic interactions of KaiA with KaiC on sub-second timescales. KaiA transiently interacts with KaiC, thereby stimulating KaiC autokinase activity. As KaiC becomes progressively more phosphorylated, KaiA’s affinity for KaiC weakens, revealing a feedback of KaiC phosphostatus back onto the KaiA-binding events. These non-equilibrium interactions integrate high-frequency binding and unbinding events, thereby refining the period of the longer term oscillations. Moreover, this differential affinity phenomenon broadens the range of Kai protein stoichiometries that allow rhythmicity, explaining how the oscillation is resilient in an in vivo milieu that includes noise. Therefore, robustness of rhythmicity on a 24-h scale is explainable by molecular events occurring on a scale of sub-seconds.

Genome-wide fitness assessment during diurnal growth reveals an expanded role of the cyanobacterial circadian clock protein KaiA

The recurrent pattern of light and darkness generated by Earth's axial rotation has profoundly influenced the evolution of organisms, selecting for both biological mechanisms that respond acutely to environmental changes and circadian clocks that program physiology in anticipation of daily variations. The necessity to integrate environmental responsiveness and circadian programming is exemplified in photosynthetic organisms such as cyanobacteria, which depend on light-driven photochemical processes. The cyanobacterium Synechococcus elongatus PCC 7942 is an excellent model system for dissecting these entwined mechanisms. Its core circadian oscillator, consisting of three proteins, KaiA, KaiB, and KaiC, transmits time-of-day signals to clock-output proteins, which reciprocally regulate global transcription. Research performed under constant light facilitates analysis of intrinsic cycles separately from direct environmental responses but does not provide insight into how these regulatory systems are integrated during light-dark cycles. Thus, we sought to identify genes that are specifically necessary in a day-night environment. We screened a dense bar-coded transposon library in both continuous light and daily cycling conditions and compared the fitness consequences of loss of each nonessential gene in the genome. Although the clock itself is not essential for viability in light-dark cycles, the most detrimental mutations revealed by the screen were those that disrupt KaiA. The screen broadened our understanding of light-dark survival in photosynthetic organisms, identified unforeseen clock-protein interaction dynamics, and reinforced the role of the clock as a negative regulator of a nighttime metabolic program that is essential for S. elongatus to survive in the dark.

The reversible function switching of the circadian clock protein KaiA is encoded in its structure

BackgroundCircadian rhythms are important to the evolution of organisms and human health, and recent studies proved that post-translational circadian clocks widely exist in all phyla. The circadian clock of cyanobacteria is an important model system as the first verified circadian oscillator independent of transcriptional-/translational-level regulations. This circadian oscillator consists of three proteins, KaiA, KaiB, and KaiC, in which KaiA stimulates KaiC's phosphorylation but KaiB antagonizes KaiA. Despite of intense research on the molecular mechanism of this oscillator in the last decades, the regulation mechanism of KaiA's function remains unclear. MethodsIn this study, we combined computational tools and experimental assays to study the function switching of KaiA. We adopted different strategies to re-design KaiA protein to elucidate its function switch during the circadian oscillation. ResultsWe showed that KaiA's function switch is determined by its structural dynamics, and KaiB antagonizes KaiA by switching it from an active state to an inactive state with the help of KaiC. ConclusionsThe reversible function switching of KaiA is key to the KaiABC oscillator, and the switching could be regulated by the 3-D domain swapped homo-dimer conformation of KaiA, which provides the necessary structural flexibility. General significanceOur finding updated the current knowledge on the regulation of KaiA's function. This work would deepen our understanding of the KaiABC oscillator, and should be conceptually useful in the design of artificial biological oscillators.

Genome-wide identification of major protein families of cyanobacteria and genomic insight into the circadian rhythm

ABSTRACT Cyanobacteria are among the most abundant organisms present on earth and are considered to be one of the oldest known clades. Cyanobacteria are oxygenic photosynthetic bacteria and are well known as promising renewable sources of energy; therefore, it is important to understand aspects of their genomes in detail across species. Advances in sequencing technology and the availability of several cyanobacterial genomes have provided an excellent opportunity to understand the diversity and evolution of the cyanobacterial genome. Here, we compared the genomes of 62 different phototrophic cyanobacteria. Evaluation of genetic diversity of all the cyanobacteria species studied revealed that evolution from their common ancestors was polyphyletic. In addition, the genomes were very diverse and varied among species, and significant genomic diversity was observed at the species and strain level. Overall, we identified 56 different protein families of cyanobacteria species/strains and found that they varied significantly among strains of a species. The circadian clock proteins KaiA, KaiB and KaiC (KaiABC complex proteins) of cyanobacteria were found to be present and consistent in the majority of cyanobacterial species while absent in a few others. Evolutionary analysis of the KaiABC protein complex showed that the KaiA protein has a high frequency of polymorphism, and multiple alleles were found to be present at high frequency. These results demonstrated that evolution of phosphorylation events occurred via KaiA in the KaiABC complex. Furthermore, multiple sequence alignment showed that KaiA, KaiB and KaiC proteins are highly conserved in nature. Our results provide direct information regarding the presence of different protein or protein families in cyanobacteria. The information presented here will serve as a starting point to explore the genetic diversity of cyanobacteria with the potential to play important roles in biotechnological applications.

Expression and Purification of Cyanobacterial Circadian Clock Protein KaiC and Determination of Its Auto-phosphatase Activity.

Circadian rhythms are biological processes displaying an endogenous oscillation with a period of ~24 h. They allow organisms to anticipate and get prepared for the environmental changes caused mainly by the rotation of Earth. Circadian rhythms are driven by circadian clocks that consist of proteins, DNA, and/or RNA. Circadian clocks of cyanobacteria are the simplest and one of the best studied models. They contain the three clock proteins KaiA, KaiB, and KaiC which can be used for in vitro reconstitution experiments and determination of the auto-phosphatase activity of KaiC as described in this protocol.

Site‐directed spin labeling‐electron spin resonance mapping of the residues of cyanobacterial clock protein KaiA that are affected by KaiA–KaiC interaction

The cyanobacterial clock proteins KaiA, KaiB and KaiC interact with each other to generate circadian oscillations. We have identified the residues of the KaiA homodimer affected through association with hexameric KaiC (KaiC6mer) using a spin-label-tagged KaiA C-terminal domain protein (KaiAc) and performing electron spin resonance (ESR) analysis. Cys substitution and/or the attachment of a spin label to residues located at the bottom area of the KaiAc concave surface, a KaiC-binding groove, hindered the association of KaiAc with KaiC6mer, suggesting that the groove likely mediates the interaction with KaiC6mer. The residues affected by KaiC6mer association were concentrated in the three areas: the concave surface, a lobe-like structure (a mobile lobe near the concave surface) and a region adjacent to both the concave surface and the mobile lobe. The distance between the two E254, D255, L258 and R252 residues located on the mobile lobe decreased after KaiC association, suggesting that the two mobile lobes approach each other during the interaction. Analyzing the molecular dynamics of KaiAc showed that these structural changes suggested by ESR analysis were possible. Furthermore, the analyses identified three asymmetries in KaiAc dynamic structures, which gave us a possible explanation of an asymmetric association of KaiAc with KaiC6mer.

Hypersensitive Photic Responses and Intact Genome-Wide Transcriptional Control without the KaiC Phosphorylation Cycle in the Synechococcus Circadian System

Cyanobacteria are unique organisms with remarkably stable circadian oscillations. These are controlled by a network architecture that comprises two regulatory factors: posttranslational oscillation (PTO) and a transcription/translation feedback loop (TTFL). The clock proteins KaiA, KaiB, and KaiC are essential for the circadian rhythm of the unicellular species Synechococcus elongatus PCC 7942. Temperature-compensated autonomous cycling of KaiC phosphorylation has been proposed as the primary oscillator mechanism that maintains the circadian clock, even in the dark, and it controls genome-wide gene expression rhythms under continuous-light conditions (LL). However, the kaiC(EE) mutation (where "EE" represents the amino acid changes Ser431Glu and Thr432Glu), where phosphorylation cycling does not occur in vivo, has a damped but clear kaiBC expression rhythm with a long period. This suggests that there must be coupling between the robust PTO and the "slave" unstable TTFL. Here, we found that the kaiC(EE) mutant strain in LL was hypersensitive to the dark acclimation required for phase shifting. Twenty-three percent of the genes in the kaiC(EE) mutant strain exhibited genome-wide transcriptional rhythms with a period of 48 h in LL. The circadian phase distribution was also conserved significantly in most of the wild-type and kaiC(EE) mutant strain cycling genes, which suggests that the output mechanism was not damaged severely even in the absence of KaiC phosphorylation cycles. These results strongly suggest that the KaiC phosphorylation cycle is not essential for generating the genome-wide rhythm under light conditions, whereas it is important for appropriate circadian timing in the light and dark.

3P040 藍色細菌時計タンパク質KaiA-KaiC相互作用のESR解析(01B.蛋白質:構造機能相関,ポスター,日本生物物理学会年会第51回(2013年度))

3P040 藍色細菌時計タンパク質KaiA-KaiC相互作用のESR解析(01B.蛋白質:構造機能相関,ポスター,日本生物物理学会年会第51回(2013年度))

3B1046 時計タンパク質KaiA-KaiC相互作用のESR解析(蛋白質-構造機能相関III:動態,生体リズム,口頭発表,日本生物物理学会第50回年会(2012年度))

3B1046 時計タンパク質KaiA-KaiC相互作用のESR解析(蛋白質-構造機能相関III:動態,生体リズム,口頭発表,日本生物物理学会第50回年会(2012年度))

2P316 Specific residues involved in interaction between cyanobacterial clock protein KaiA and KaiC revealed by SDSL-ESR analysis(The 48th Annual Meeting of the Biophysical Society of Japan)

2P316 Specific residues involved in interaction between cyanobacterial clock protein KaiA and KaiC revealed by SDSL-ESR analysis(The 48th Annual Meeting of the Biophysical Society of Japan)

1P-250 ESR法を用いたシアノバクテリア時計タンパク質KaiA-KaiC相互作用の解析(非平衡・生体リズム,第47回日本生物物理学会年会)

1P-250 ESR法を用いたシアノバクテリア時計タンパク質KaiA-KaiC相互作用の解析(非平衡・生体リズム,第47回日本生物物理学会年会)

Regulation of Circadian Clock Gene Expression by Phosphorylation States of KaiC in Cyanobacteria

Three clock proteins--KaiA, KaiB, and KaiC--have been identified as essential components of the circadian oscillator in cyanobacteria, and Kai-based chemical oscillation is thought to be the basic circadian timing mechanism in Synechococcus elongatus PCC 7942. Transcription and translation of kaiBC in cyanobacterial cells was quantitatively studied to elucidate how these processes are coupled to the chemical oscillator using a strain in which circadian oscillation is under the control of IPTG (isopropyl-beta-D-thiogalactopyranoside). The kinetics of repression of kaiBC promoter triggered by IPTG allowed estimation of transient response at 10 h. This response time is suitable for cyanobacterial transcription and/or translation to match with the Kai-based oscillator. Interestingly, kaiBC promoter activity and KaiC phosphorylation showed robust circadian rhythms, whereas trc promoter-driven kaiBC mRNA levels and KaiC accumulation were almost arrhythmic. These results indicate that cyanobacterial circadian rhythms can be generated even if kaiBC expression is constitutive. Moreover, there was a positive correlation between activation of the kaiBC promoter and an increase in the KaiC phosphorylation ratio in three rhythmic conditions. Based on these observations, it is likely that the KaiC phosphorylation ratio is the main factor in the activation of kaiBC promoter. Finally, we quantitatively compared the threshold level of phosphorylated KaiC for the repression or derepression of kaiBC promoter and found that this parameter is an important factor in repressing the kaiBC promoter.

An allosteric model of circadian KaiC phosphorylation

In a recent series of ground-breaking experiments, Nakajima et al. [Nakajima M, Imai K, Ito H, Nishiwaki T, Murayama Y, Iwasaki H, Oyama T, Kondo T (2005) Science 308:414-415] showed that the three cyanobacterial clock proteins KaiA, KaiB, and KaiC are sufficient in vitro to generate circadian phosphorylation of KaiC. Here, we present a mathematical model of the Kai system. At its heart is the assumption that KaiC can exist in two conformational states, one favoring phosphorylation and the other dephosphorylation. Each individual KaiC hexamer then has a propensity to be phosphorylated in a cyclic manner. To generate macroscopic oscillations, however, the phosphorylation cycles of the different hexamers must be synchronized. We propose a novel synchronization mechanism based on differential affinity: KaiA stimulates KaiC phosphorylation, but the limited supply of KaiA dimers binds preferentially to those KaiC hexamers that are falling behind in the oscillation. KaiB sequesters KaiA and stabilizes the dephosphorylating KaiC state. We show that our model can reproduce a wide range of published data, including the observed insensitivity of the oscillation period to variations in temperature, and that it makes nontrivial predictions about the effects of varying the concentrations of the Kai proteins.

A Model for the Circadian Rhythm of Cyanobacteria that Maintains Oscillation without Gene Expression

An intriguing property of the cyanobacterial circadian clock is that endogenous rhythm persists when protein abundances are kept constant either in the presence of translation and transcription inhibitors or in the constant dark condition. Here we propose a regulatory mechanism of KaiC phosphorylation for the generation of circadian oscillations in cyanobacteria. In the model, clock proteins KaiA and KaiB are assumed to have multiple states, regulating the KaiC phosphorylation process. The model can explain 1), the sustained oscillation of gene expression and protein abundance when the expression of the kaiBC gene is regulated by KaiC protein, and 2), the sustained oscillation of phosphorylated KaiC when transcription and translation processes are inhibited and total protein abundance is fixed. Results of this work suggest that KaiA and KaiB strengthen the nonlinearity of KaiC phosphorylation, thereby promoting the circadian rhythm in cyanobacteria.

Structure of the C-terminal domain of the clock protein KaiA in complex with a KaiC-derived peptide: implications for KaiC regulation.

Circadian clocks are widespread endogenous mechanisms that control the temporal pattern of diverse biological processes, including gene transcription. KaiA is the positive element of the cyanobacterial clock because KaiA overexpression elevates transcription levels of clock components. Recently, we showed that the structure of KaiA is that of a domain-swapped homodimer. The N-terminal domain is a pseudo-receiver; thus, it is likely to be involved in signal transduction in the clock-resetting pathway. The C-terminal domain of KaiA is structurally novel and enhances the KaiC autokinase activity directly. Here, we report the NMR structure of the C-terminal domain of KaiA (ThKaiA180C) in complex with a KaiC-derived peptide from the cyanobacterium Thermosynechococcus elongatus BP-1. The protein-peptide interface is revealed to be different from a model that was proposed earlier, is stabilized by a combination of hydrophobic and electrostatic interactions, and includes many residues known to produce a circadian-period phenotype upon substitution. Although the structure of the monomeric subunit of ThKaiA180C is largely unchanged upon peptide binding, the intersubunit dimerization angle changes. It is proposed that modulation of the C-terminal KaiA domain dimerization angle regulates KaiA-KaiC interactions.

Anabaena circadian clock proteins KaiA and KaiB reveal a potential common binding site to their partner KaiC.

The cyanobacterial clock proteins KaiA and KaiB are proposed as regulators of the circadian rhythm in cyanobacteria. Mutations in both proteins have been reported to alter or abolish circadian rhythmicity. Here, we present molecular models of both KaiA and KaiB from the cyanobacteria Anabaena sp PCC7120 deduced by crystal structure analysis, and we discuss how clock-changing or abolishing mutations may cause their resulting circadian phenotype. The overall fold of the KaiA monomer is that of a four-helix bundle. KaiB, on the other hand, adopts an alpha-beta meander motif. Both proteins purify and crystallize as dimers. While the folds of the two proteins are clearly different, their size and some surface features of the physiologically relevant dimers are very similar. Notably, the functionally relevant residues Arg 69 of KaiA and Arg 23 of KaiB align well in space. The apparent structural similarities suggest that KaiA and KaiB may compete for a potential common binding site on KaiC.