Tatsuya Uzumaki

ATP-induced hexameric ring structure of the cyanobacterial circadian clock protein KaiC.

Background: KaiA, KaiB and KaiC are cyanobacterial circadian clock proteins. KaiC contains two ATP/GTP‐binding Walkers motif As, and mutations in these regions affect the clock oscillations.

Crystal structure of the C-terminal clock-oscillator domain of the cyanobacterial KaiA protein

KaiA, KaiB and KaiC constitute the circadian clock machinery in cyanobacteria, and KaiA activates kaiBC expression whereas KaiC represses it. Here we show that KaiA is composed of three functional domains, the N-terminal amplitude-amplifier domain, the central period-adjuster domain and the C-terminal clock-oscillator domain. The C-terminal domain is responsible for dimer formation, binding to KaiC, enhancing KaiC phosphorylation and generating the circadian oscillations. The X-ray crystal structure at a resolution of 1.8 Å of the C-terminal clock-oscillator domain of KaiA from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 shows that residue His270, located at the center of a KaiA dimer concavity, is essential to KaiA function. KaiA binding to KaiC probably occurs via the concave surface. On the basis of the structure, we predict the structural roles of the residues that affect circadian oscillations.

Functionally important substructures of circadian clock protein KaiB in a unique tetramer complex.

KaiB is a component of the circadian clock molecular machinery in cyanobacteria, which are the simplest organisms that exhibit circadian rhythms. Here we report the x-ray crystal structure of KaiB from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. The KaiB crystal diffracts at a resolution of 2.6 Å and includes four subunits organized as a dimer of dimers, each composed of two non-equivalent subunits. The overall shape of the tetramer is an elongated hexagonal plate, with a single positively charged cleft flanked by two negatively charged ridges whose surfaces includes several terminal chains. Site-directed mutagenesis of Synechococcus KaiB confirmed that alanine substitution of residues Lys-11 or Lys-43 in the cleft, or deletion of C-terminal residues 95–108, which forms part of the ridges, strongly weakens in vivo circadian rhythms. Characteristics of KaiB deduced from the x-ray crystal structure were also confirmed by physicochemical measurements of KaiB in solution. These data suggest that the positively charged cleft and flanking negatively charged ridges in KaiB are essential for the biological function of KaiB in the circadian molecular machinery in cyanobacteria.

ATPase activity and its temperature compensation of the cyanobacterial clock protein KaiC

KaiA, KaiB and KaiC constitute the circadian clock machinery in cyanobacteria. KaiC is a homohexamer; its subunit contains duplicated halves, each with a set of ATPase motifs. Here, using highly purified KaiC preparations of the thermophilic cyanobacterium Thermosynechococcus elongatus BP‐1 produced in Escherichia coli, we found that the N‐ and C‐terminal domains of KaiC had extremely weak ATPase activity. ATPase activity showed temperature compensation in wild‐type KaiC, but not in KaiCS431A/T432A, a mutant that lacks two phosphorylation sites. We concluded that KaiC phosphorylation is involved in the ATPase temperature‐compensation mechanism—which is probably critical to the stability of the circadian clock in cyanobacteria—and we hypothesized the following temperature‐compensation mechanism: (i) The C‐terminal phosphorylation sites of a KaiC hexamer subunit are phosphorylated by the C‐terminal domain of an adjacent KaiC subunit; (ii) the phosphorylation suppresses the ATPase activity of the C‐terminal domain; and (iii) the phosphorylated KaiC spontaneously dephosphorylates, resulting in the recover of ATPase activity.

The roles of the dimeric and tetrameric structures of the clock protein KaiB in the generation of circadian oscillations in cyanobacteria

Background: The function of KaiB remains to be solved. Results: Dimeric KaiB1–94 generated circadian oscillation in vitro, but it did not in cells. Conclusion: KaiB tetramer-dimer transformation is responsible for the regulation of the SasA-mediated clock output pathway. Significance: We demonstrated the role of KaiB in the regulation of the SasA-KaiC interaction, involved in the transmission of time-information from KaiABC-machinery to transcription apparatus. The molecular machinery of the cyanobacterial circadian clock consists of three proteins, KaiA, KaiB, and KaiC. The three Kai proteins interact with each other and generate circadian oscillations in vitro in the presence of ATP (an in vitro KaiABC clock system). KaiB consists of four subunits organized as a dimer of dimers, and its overall shape is that of an elongated hexagonal plate with a positively charged cleft flanked by two negatively charged ridges. We found that a mutant KaiB with a C-terminal deletion (KaiB1–94), which lacks the negatively charged ridges, was a dimer. Despite its dimeric structure, KaiB1–94 interacted with KaiC and generated normal circadian oscillations in the in vitro KaiABC clock system. KaiB1–94 also generated circadian oscillations in cyanobacterial cells, but they were weak, indicating that the C-terminal region and tetrameric structure of KaiB are necessary for the generation of normal gene expression rhythms in vivo. KaiB1–94 showed the highest affinity for KaiC among the KaiC-binding proteins we examined and inhibited KaiC from forming a complex with SasA, which is involved in the main output pathway from the KaiABC clock oscillator in transcription regulation. This defect explains the mechanism underlying the lack of normal gene expression rhythms in cells expressing KaiB1–94.

The Circadian Clock-Related Gene pex Regulates a Negative cis Element in the kaiA Promoter Region

In the cyanobacterium Synechococcus sp. strain PCC 7942, a circadian clock-related gene, pex, was identified as the gene prolonging the period of the clock. A PadR domain, which is a newly classified transcription factor domain, and the X-ray crystal structure of the Pex protein suggest a role for Pex in transcriptional regulation in the circadian system. However, the regulatory target of the Pex protein is unknown. To determine the role of Pex, we monitored bioluminescence rhythms that reported the expression activity of the kaiA gene or the kaiBC operon in pex deficiency, pex constitutive expression, and the wild-type genotype. The expression of kaiA in the pex-deficient or constitutive expression genotype was 7 or 1/7 times that of the wild type, respectively, suggesting that kaiA is the target of negative regulation by Pex. In contrast, the expression of the kaiBC gene in the two pex-related genotypes was the same as that in the wild type, suggesting that Pex specifically regulates kaiA expression. We used primer extension analysis to map the transcription start site for the kaiA gene 66 bp upstream of the translation start codon. Mapping with deletion and base pair substitution of the kaiA upstream region revealed that a 5-bp sequence in this region was essential for the regulation of kaiA. The repression or constitutive expression of the kaiA transgene caused the prolongation or shortening of the circadian period, respectively, suggesting that the Pex protein changes the period via the negative regulation of kaiA.

Direct interaction between KaiA and KaiB revealed by a site-directed spin labeling electron spin resonance analysis.

In cyanobacteria, three clock proteins, KaiA, KaiB and KaiC, play essential roles in generating circadian oscillations. The interactions of these proteins change during the circadian cycle. Here, we demonstrated direct interaction between KaiA and KaiB using electron spin resonance spectroscopy. We prepared cystein (Cys)‐substituted mutants of Thermosynechococcus elongatus KaiB, labeled specifically their Cys residues with spin labels and measured the ESR spectra of the labeled KaiB. We found that KaiB labeled at the 64th residue showed spectral changes in the presence of KaiA, but not in the presence of KaiC or bovine serum albumin as a negative control. KaiB labeled at the 101st residue showed no such spectral changes even in the presence of KaiA. The results suggest that KaiB interacts with KaiA in the vicinity of the 64th residue of KaiB. Further analysis demonstrated that the C‐terminal clock‐oscillator domain of KaiA is responsible for this interaction.

Functionally important structural elements of the cyanobacterial clock-related protein Pex

Pex, a clock‐related protein involved in the input pathway of the cyanobacterial circadian clock system, suppresses the expression of clock gene kaiA and lengthens the circadian period. Here, we determined the crystal structure of Anabaena Pex (AnaPex; Anabaena sp. strain PCC 7120) and Synechococcus Pex (SynPex; Synechococcus sp. strain PCC 7942). Pex is a homodimer that forms a winged‐helix structure. Using the DNase I protection and electrophoresis mobility shift assays on a Synechococcus kaiA upstream region, we identified a minimal 25‐bp sequence that contained an imperfectly inverted repeat sequence as the Pex‐binding sequence. Based on crystal structure, we predicted the amino acid residues essential for Pexs DNA‐binding activity and examined the effects of various Ala‐substitutions in the α3 helix and wing region of Pex on in vitro DNA‐binding activity and in vivo rhythm functions. Mutant AnaPex proteins carrying a substitution in the wing region displayed no specific DNA‐binding activity, whereas those carrying a substitution in the α3 helix did display specific binding activity. But the latter were less thermostable than wild‐type AnaPex and their in vitro functions were defective. We concluded that Pex binds a kaiA upstream DNA sequence via its wing region and that its α3 helix is probably important to its stability.

Crystallization and preliminary crystallographic analysis of the circadian clock protein KaiB from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1

KaiB is a component of the circadian clock oscillator in cyanobacteria, which are the simplest organisms that exhibit circadian rhythms. KaiB consists of 108 amino-acid residues and has a molecular weight of 12 025 Da. KaiB and Cys-substituted KaiB mutants from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 were expressed as GST-fusion proteins in Escherichia coli, purified and crystallized. The crystals of wild-type KaiB belong to the monoclinic space group P2(1), with unit-cell parameters a = 89.6, b = 71.2, c = 106.8 A, beta = 100.1 degrees. While the native crystals diffract to 3.7 A, osmium derivatives, which show an approximately 4 A shrinkage in the b axis, diffract to 2.6 A. The crystals of the singly Cys-substituted mutant T64C with Hg, which show different morphology, diffract to 2.5 A and belong to the monoclinic space group P2, with unit-cell parameters a = 63.7, b = 33.4, c = 93.7 A, beta = 100.1 degrees. Anomalous difference Patterson maps of the Os- and Hg-derivative crystals had significant peaks in their Harker sections, suggesting that both derivatives are suitable for structure determination.

Unidirectional Electron Transfer in Chlorophyll d -Containing Photosystem I Reaction Center Complex of Acaryochloris marina

The purified photosystem I (PS I) reaction center complex of a cyanobacterium Acaryochloris marina contained 88 Chl d: 1.1 Chl a: 19 carotenoids: 2.0 phylloquinone. Amino acid sequences of PsaA and PsaB polypeptides were almost homologous to those in the other cyanobacteria. The ligands for A0 was Met698A and that for A0′ was Leu688B but not Met that is conserved in all the other PS I. Laser excitation induced the 10-ps bleach and the 40- ps recovery at 680 nm of Chl a-type pigment in parallel with the 49-ps bleach of the Chl d-dimer P740 at 740 nm. The results indicate that A0 is Chl a-680 ligated by Met698A and that the PsaB branch with Leu688B is inactive for the electron transfer. The PS I of A. marina, thus, is the unique asymmetric type I reaction center with the unidirectional electron transfer pathway only through PsaA branch.