Stoichiometric interactions between cyanobacterial clock proteins KaiA and KaiC

Hayashi, Fumio; Ito, Hiroki; Fujita, Masayasu; Iwase, Ryo; Uzumaki, Tatsuya; Ishiura, Masahiro

doi:10.1016/j.bbrc.2004.02.034

Cited by 51 publications

(106 citation statements)

References 19 publications

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“…Arrows indicate peaks for free KaiC-EE497 (red peak) and the complex formed by KaiA-130C, KaiB, and KaiC-EE497 (black peak). The apparent molecular size of the complex (approximately 440 kDa) was close to the size expected for a complex composed of a KaiC-EE497 hexamer, two KaiA-130C dimers, and two KaiB dimers (460 kDa) (27,55). (C) SDS-PAGE gel of the elution peak indicated by the black arrow in B.…”

Section: Kaicb(a) Is Formed By Direct Interactions With the Linker Sementioning

confidence: 53%

Flexibility of the C-terminal, or CII, ring of KaiC governs the rhythm of the circadian clock of cyanobacteria

Chang

Kuo

Tseng

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

144

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In the cyanobacterial circadian oscillator, KaiA and KaiB alternately stimulate autophosphorylation and autodephosphorylation of KaiC with a periodicity of approximately 24 h. KaiA activates autophosphorylation by selectively capturing the A loops of KaiC in their exposed positions. The A loops and sites of phosphorylation, residues S431 and T432, are located in the CII ring of KaiC. We find that the flexibility of the CII ring governs the rhythm of KaiC autophosphorylation and autodephosphorylation and is an example of dynamics-driven protein allostery. KaiA-induced autophosphorylation requires flexibility of the CII ring. In contrast, rigidity is required for KaiC-KaiB binding, which induces a conformational change in KaiB that enables it to sequester KaiA by binding to KaiA's linker. Autophosphorylation of the S431 residues around the CII ring stabilizes the CII ring, making it rigid. In contrast, autophosphorylation of the T432 residues offsets phospho-S431-induced rigidity to some extent. In the presence of KaiA and KaiB, the dynamic states of the CII ring of KaiC executes the following circadian rhythm: CII ST flexible → CII SpT flexible → CII pSpT rigid → CII pST very-rigid → CII ST flexible . Apparently, these dynamic states govern the pattern of phosphorylation, ST → SpT → pSpT → pST → ST. CII-CI ring-on-ring stacking is observed when the CII ring is rigid, suggesting a mechanism through which the ATPase activity of the CI ring is rhythmically controlled. SasA, a circadian clock-output protein, binds to the CI ring. Thus, rhythmic ring stacking may also control clock-output pathways.

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Section: Kaicb(a) Is Formed By Direct Interactions With the Linker Sementioning

confidence: 53%

Flexibility of the C-terminal, or CII, ring of KaiC governs the rhythm of the circadian clock of cyanobacteria

Chang

Kuo

Tseng

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

144

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“…KaiC abundance in vivo is relatively high compared with KaiA (19), and therefore, we expect that a KaiA dimer will be bound to two KaiC protein molecules for at least some fraction of the circadian period. However, KaiA can fully enhance the KaiC autokinase activity even at relatively low KaiA͞KaiC stoichiometries (34), suggesting that formation of the KaiA-KaiC complex is transient. Currently, it is not known whether the KaiC autokinase activity operates as cis or trans (transphosphorylation).…”

Section: Discussionmentioning

confidence: 99%

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

Vakonakis

LiWang

2004

Proc. Natl. Acad. Sci. U.S.A.

103

142

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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.

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“…In the presence of ATP, KaiC is phosphorylated by KaiC itself, and the phosphorylation is enhanced by KaiA (7)(8)(9)(10). We demonstrated that two molecules of KaiA dimer can interact with one molecule of KaiC hexamer and that one molecule of KaiA dimer interacting with one molecule of KaiC hexamer is enough to enhance KaiC phosphorylation (5). By x-ray crystal structure analysis, in vitro biochemical analysis, and in vivo rhythm assays, we demonstrated that the His 270 residue in the C-terminal clock-oscillator domain of KaiA is essential to clock oscillation in vivo and important for both the binding of KaiA to KaiC and the enhancement of KaiC phosphorylation by KaiA (7).…”

mentioning

confidence: 82%

“…Assay for the Phosphorylation of KaiCs in the Presence or Absence of KaiA-We assayed KaiC phosphorylation as described previously (5). Briefly, KaiC monomer (7.3 pmol in hexamer) was incubated with 1 mM ATP in 30 l of 20 mM Tris-HCl buffer (pH 7.5) containing 5 mM MgCl 2 at 50°C for various times in the presence or absence of 43.8 pmol of KaiA (in dimer).…”

Section: Methodsmentioning

confidence: 99%

“…Production of KaiCs in E. coli and Their Purification-Production of KaiCs (KaiC WT , KaiC K53H , KaiC K294H , KaiC CatE1 Ϫ, and KaiC CatE2 Ϫ) in E. coli BL21, their purification, protein determination, and SDS-PAGE were carried out as described previously (5). Briefly, KaiCs were overproduced in E. coli cells as soluble GST fusion proteins and trapped with glutathione-Sepharose-4B.…”

Section: Methodsmentioning

confidence: 99%

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Roles of Two ATPase-Motif-containing Domains in Cyanobacterial Circadian Clock Protein KaiC

Hayashi

Itoh

Uzumaki

et al. 2004

Journal of Biological Chemistry

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Cyanobacterial clock protein KaiC has a hexagonal, pot-shaped structure composed of six identical dumbbell-shaped subunits. Each subunit has duplicated domains, and each domain has a set of ATPase motifs. The two spherical regions of the dumbbell are likely to correspond to two domains. We examined the role of the two sets of ATPase motifs by analyzing the in vitro activity of ATP␥S binding, AMPPNP-induced hexamerization, thermostability, and phosphorylation of KaiC and by in vivo rhythm assays both in wild type KaiC (KaiC WT ) and KaiCs carrying mutations in either Walker motif A or deduced catalytic Glu residues. We demonstrated that 1) the KaiC subunit had two types of ATPbinding sites, a high affinity site in N-terminal ATPase motifs and a low affinity site in C-terminal ATPase motifs, 2) the N-terminal motifs were responsible for hexamerization, and 3) the C-terminal motifs were responsible for both stabilization and phosphorylation of the KaiC hexamer. We proposed the following reaction mechanism. ATP preferentially binds to the N-terminal high affinity site, inducing the hexamerization of KaiC. Additional ATP then binds to the C-terminal low affinity site, stabilizing and phosphorylating the hexamer. We discussed the effect of these KaiC mutations on circadian bioluminescence rhythm in cells of cyanobacteria.Circadian rhythms, 24-h biological oscillations of metabolic and behavioral activities observed ubiquitously in prokaryotes and eukaryotes, are endogenously regulated by circadian clocks. Several clock and clock-related genes from Drosophila, Neurospora, Arabidopsis, mice, and cyanobacteria have been cloned and analyzed (1).Cyanobacteria are the simplest organisms that exhibit circadian rhythms. We previously cloned and analyzed the kaiABC circadian clock gene cluster in the cyanobacterium Synechococcus sp. strain PCC 7942 (hereafter called Synechococcus) that is essential for the generation of circadian rhythms in cyanobacteria. The gene cluster consists of two

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Stoichiometric interactions between cyanobacterial clock proteins KaiA and KaiC

Cited by 51 publications

References 19 publications

Flexibility of the C-terminal, or CII, ring of KaiC governs the rhythm of the circadian clock of cyanobacteria

Flexibility of the C-terminal, or CII, ring of KaiC governs the rhythm of the circadian clock of cyanobacteria

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

Roles of Two ATPase-Motif-containing Domains in Cyanobacterial Circadian Clock Protein KaiC

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