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 KaiB_1–94 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.

Abstract

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 (KaiB_1–94), which lacks the negatively charged ridges, was a dimer. Despite its dimeric structure, KaiB_1–94 interacted with KaiC and generated normal circadian oscillations in the in vitro KaiABC clock system. KaiB_1–94 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. KaiB_1–94 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 KaiB_1–94.

Introduction

The circadian clock is an endogenous biological mechanism that generates daily cycles in physiological activity (circadian rhythms) (1, 2). Cyanobacteria are the simplest organisms that exhibit circadian rhythms (2), and the circadian clock gene cluster kaiABC is essential for circadian rhythms in cyanobacteria (3). The rhythms involve circadian oscillations in the phosphorylation level (4) and ATPase activity (5) of KaiC and complex formation among KaiA, KaiB, and KaiC (6, 7). KaiC has a duplicated RecA/DnaB structure. Its N-terminal domain shows ATPase activity (8), whereas its C-terminal domain shows both ATPase activity (5, 8) and intersubunit phosphorylation activity (9, 10). KaiA and KaiB have opposite effects on KaiC ATPase activity (5, 8) and phosphorylation level; KaiA increases them (10–13), and KaiB decreases them (5, 12, 14). KaiB is composed of two asymmetric dimers (15, 16) that form a tetramer. KaiB directly associates with the C-terminal clock oscillator domain (13) of KaiA (17). Its overall shape is that of an elongated hexagonal plate with a positively charged cleft (PC)⁷ that is flanked by two negatively charged ridges (NRs) (16). Rhythm analysis of cyanobacterial cells carrying a mutant kaiB gene show that the PC is necessary to KaiB clock function (16). Deletion of the C-terminal residues from amino acids 95–108 of KaiB (yielding mutant KaiB_1–94) derived from the cyanobacteria Synechococcus sp. strain PCC 7942 (hereafter Synechococcus) and Thermosynechococcus elongatus results in loss of the NRs and extensively weakens in vivo circadian rhythms (16). Here we examine the structure of KaiB_1–94 and the role it plays in the generation circadian oscillations.

EXPERIMENTAL PROCEDURES

Protein Expression and Purification

KaiA, KaiB, KaiC, and SasA derived from T. elongatus (18) were expressed in Escherichia coli BL21 cells and purified as previously described (16, 17, 19–21). We generated gene constructs for a truncated mutant KaiB with a C-terminal deletion from residues 95–108 (KaiB_1–94) and a mutant KaiC with alanine (KaiC_AA) or aspartate (KaiC_DD) substitutions at the two KaiC phosphorylation sites using PCR-mediated site-directed mutagenesis and cloned them in the pGEX-6P-1 vector (GE Healthcare) as described previously (20).

To determine their purity, we subjected the purified proteins to SDS-PAGE on 15% gels (22) and stained the gels with Coomassie Brilliant Blue (CBB). We estimated protein concentrations using the Bio-Rad Protein Assay with BSA as the standard as previously described (20). Unless otherwise stated, KaiA, KaiB, KaiC, and SasA refer to the KaiA dimer, KaiB tetramer, KaiC hexamer, and SasA trimer (21), respectively, and their concentrations are expressed in terms of their oligomeric status.

Estimation of the Molecular Mass of KaiB by Gel Filtration Chromatography

We estimated the molecular mass of KaiB using analytical gel filtration chromatography on a Superdex 75 5/150GL column (GE Healthcare) equilibrated with 20 mm Tris-HCl buffer (pH 7.5) containing 150 mm NaCl (gel filtration buffer) at 4 °C using an ÄKTA explorer (GE Healthcare) and a Gel Filtration Calibration Kit LMW (GE Healthcare) for the molecular mass standards. We monitored the elution profiles of proteins by absorbance at 280 nm (A₂₈₀) and performed all analyses in 20 mm Tris-HCl buffer (pH 7.5) containing 50 mm NaCl, 20 mm Tris-HCl buffer (pH 7.5) containing 500 mm NaCl, 20 mm MES-NaOH buffer (pH 6.5) containing 150 mm NaCl, and 20 mm Tris-HCl buffer (pH 8.5) containing 150 mm NaCl.

Sedimentation Equilibrium by Analytical Ultracentrifugation

We performed sedimentation equilibrium analytical ultracentrifugation using a Beckman Optima XL-A analytical ultracentrifuge with an An60Ti rotor. Samples were dialyzed against 20 mm HEPES-NaOH buffer (pH 7.5) containing 150 mm NaCl (sedimentation equilibrium buffer), which was used as the blank. We performed measurements at 20 °C at 36,000, 38,000, and 40,000 rpm for KaiB_1–94 and at 20,000, 22,000, and 24,000 rpm for wild-type KaiB (KaiB_WT). We monitored concentration profiles of the samples by A₂₈₀ and recorded them at a spacing of 0.001 cm in step mode, with 20 averages per step. We analyzed equilibrium data with Beckman Optima XL-A/XL-I data analyses software, Version 6.04, which was provided as an add-on to Version 6.0 (Microcal Inc.), and we calculated global, single-species fits using different loading absorbance values at 280 nm (0.2, 0.25, and 0.3 for KaiB_1–94 and 0.18 for KaiB_WT). Based on the amino acid compositions of the proteins, we used the partial specific volumes 0.767 ml/g for KaiB_1–94 and 0.759 ml/g for KaiB_WT for the analyses.

Estimation of the Molecular Mass and Stoichiometry of the KaiB-KaiC Complex by Gel Filtration Chromatography

Reaction mixtures containing 5 μm KaiB_1–94 or 2.5 μm KaiB_WT and 1 μm KaiC_DD in 20 mm HEPES-NaOH buffer (pH 7.5) containing 1 mm ATP, 5 mm MgCl₂, and 150 mm NaCl (HEPES reaction buffer) were incubated at 40 °C for 18 h and analyzed by gel filtration chromatography on a Superdex 200/HR 10/300 column (GE Healthcare) equilibrated with HEPES reaction buffer containing 0.1 mm ATP and 5 mm MgCl₂ at 4 °C. We used thyroglobulin (670 kDa), apoferritin (440 kDa), β-amylase (200 kDa), and BSA (66 kDa) as molecular mass standards. Fractions containing the KaiB-KaiC_DD complex were subjected to SDS-PAGE on 18% gels, and then the gels were stained with CBB. We determined the amount of KaiB and KaiC_DD contained in the complex by densitometry using a CS analyzer (ATTO) to determine the stoichiometry of the complex.

Labeling of KaiB with Cy3 and Fluorescence Correlation Spectroscopy (FCS) Measurements

Cy3-NHS fluorescent dye esters (GE Healthcare) were covalently coupled to the amines of lysine residues and the N-terminal amino acid residue of KaiB. KaiB (8 μm KaiB_1–94 or 4 μm KaiB_WT) was incubated with 80 μm Cy3-NHS ester in gel filtration buffer at 4 °C for 2 h. After labeling, the reaction mixtures were loaded on a PD MidiTrap^TM G-25 column (GE Healthcare) for removal of the remaining unbound dye from the labeled proteins. We calculated the amounts of Cy3 introduced onto KaiB from the absorbance of the labeled proteins at 552 nm (A₅₅₂). Under these conditions, 0.85 ± 0.10 and 0.64 ± 0.01 molecules of Cy3 (n = 3) per subunit of KaiB_1–94 and KaiB_WT were introduced into KaiB_1–94 and KaiB_WT, respectively.

We performed FCS measurements using a multiphoton FCS/fluorescence cross-correlation spectroscopy system using Fluctuation Dual Emission Uni-laser eXcitation (Fluc DEUX^TM) (MBL, Nagoya, Japan). Cy3 was excited by the 445-nm laser, and its emission was detected at 615–690 nm. Cy3-KaiB_1–94 and Cy3-KaiB_WT (0.1 μm) were separately incubated with 0, 0.03, 0.05, 0.10, and 0.30 μm KaiC_DD in the presence of 0, 0.05, 0.10, or 0.30 μm SasA in HEPES reaction buffer at 25 °C for 18 h, and the reaction mixtures were subjected to FCS measurements at 25 °C for 5 s (5 times consecutively). The diffusion time defined as the average time required for the diffusion of fluorescent particles across the detection area reflects the size of the particles. If the effect of the molecular shape on its diffusion is negligible, diffusion time is proportional to the cubic root of the molecular mass, as described in Equation 1 (23, 24),

where DT_a and DT_b are the diffusion times of molecules a (Cy3-KaiB_1–94 or Cy3-KaiB_WT) and b (Cy3-KaiB_1–94-KaiC_DD complex or Cy3-KaiB_WT-KaiC_DD), respectively, and MM_a and MM_b are the molecular masses of molecules a and b, respectively. Accordingly, we calculated the size of the Cy3-KaiB_1–94-KaiC_DD and Cy3-KaiB_WT-KaiC_DD complexes to be 320 ± 70 and 370 ± 90 kDa, respectively, using the molecular masses of Cy3-KaiB_WT (∼50 kDa) and Cy3-KaiB_1–94 (∼25 kDa). These values are consistent with the molecular masses determined by gel filtration chromatography (see Fig. 2A).

FIGURE 2.

Stoichiometry of the KaiB-KaiC complex and the formation of the KaiB-KaiC complex analyzed by FCS analysis. A, shown are elution profiles. Reaction mixtures containing 5 μm KaiB_1–94 or 2.5 μm KaiB_WT and 1 μm KaiC_DD were incubated at 40 °C for 18 h in HEPES reaction buffer and then subjected to gel filtration chromatography. Black line, KaiB_1–94 + KaiC_DD; gray line, KaiB_WT + KaiC_DD; light gray line, KaiC_DD; black dotted line, KaiB_1–94; gray dotted line, KaiB_WT. mAu, milliabsorbance units. B, shown is SDS-PAGE. Aliquots of a fraction containing the KaiB_1–94-KaiC_DD complex or the KaiB_WT-KaiC_DD complex were subjected to SDS-PAGE. Standard amounts of KaiB_1–94, KaiB_WT, and KaiC_DD were also applied to the gels. Only the SDS-PAGE gel of the KaiB_1–94-KaiC_DD complex is shown. Lanes 1–3, triplicate samples from the fraction containing the KaiB_1–94-KaiC_DD complex; lanes 4–9 contain 5–40 pmol of KaiB_1–94; lanes 10–15 contain 5–40 pmol of KaiC_DD. C, shown are calibration curves for the KaiB_1–94-KaiC_DD complex. The relative intensities of the bands were plotted against known amounts of KaiB_1–94 and KaiC_DD, and the KaiB_1–94 and KaiC_DD bands from the KaiB_1–94-KaiC_DD complex were plotted. Typical plots from three independent experiments are shown. Open circles, known amounts of KaiB_1–94; open triangles, known amounts of KaiC_DD; closed circles and closed triangles, KaiB_1–94 and KaiC_DD in the complex, respectively. D, calibration curves for the KaiB_WT-KaiC_DD complex are shown. The relative intensities of the bands were plotted against known amounts of KaiB_WT and KaiC_DD, and the KaiB_WT and KaiC_DD bands from the KaiB_WT-KaiC_DD complex were plotted. Typical plots from three independent experiments are shown. Open circles, known amounts of KaiB_WT; open triangles, known amounts of KaiC_DD; closed circles and closed triangles, KaiB_WT and KaiC_DD in the complex, respectively. E, stoichiometry of the KaiB-KaiC_DD complex is shown. The number of molecules of KaiB_1–94 or KaiB_WT monomer per molecule of KaiC_DD contained in the KaiB-KaiC_DD complex was estimated from the plots shown in A–D. Values shown are the means ± S.D. F, KaiC_DD-induced changes in the diffusion times of Cy3-KaiB_1–94 and Cy3-KaiB_WT molecules are shown. Cy3-KaiB_1–94 (0.2 μm) and Cy3-KaiB_WT (0.1 μm) were separately incubated with 0, 0.025, 0.05, 0.15, and 0.3 μm KaiC_DD, and their diffusion times were measured by FCS analysis. Values shown are the means ± S.D. from quadruple measurements. Open circles, Cy3-KaiB_1–94; gray circles, Cy3-KaiB_WT. G, KaiC_DD-induced changes in the counts per particle for the Cy3-KaiB_1–94 and Cy3-KaiB_WT molecules are shown. The counts per particle were calculated from the data shown in F.

Surface Plasmon Resonance Analysis of Interaction between KaiB and KaiC

We analyzed the interaction between KaiB_1–94 and KaiC_DD and between KaiB_WT and KaiC_DD at 25 °C by surface plasmon resonance (25) using a Biacore and its associated software (GE Healthcare). KaiC_DD (10 μm) was immobilized on a CM5 sensor chip using an amine coupling kit (GE Healthcare) in HEPES reaction buffer containing 0.1 mm ATP, 0.1 mm dithiothreitol (DTT), and 50 mm NaCl. We monitored the association to, and dissociation from, the immobilized KaiC_DD of KaiB_1–94, KaiB_WT, KaiA, and SasA continuously at stepwise concentrations of 0.1, 0.25, 0.5, 1.0, and 1.5 μm in HEPES reaction buffer. We computed the dissociation (k_d) and association (k_a) rates by fitting a 1:1 binding model to the experimental data. We calculated the value for apparent equilibrium dissociation constant (K_D) as k_d/k_a.

Measurement of the Phosphorylation Levels of KaiC

Reaction mixtures of KaiA and KaiC (each 0.5 μm) were incubated in the presence or absence of 1.0 μm KaiB_1–94 or 0.5 μm KaiB_WT in gel filtration buffer containing 1 mm ATP and 5 mm MgCl₂ at 25 or 40 °C, and 10-μl aliquots of the reaction mixtures were removed to stop the reaction at specific time intervals. The aliquots were subjected to SDS-PAGE on 12.5% gels (acrylamide:bisacrylamide = 144:1), and the gels were stained with CBB. The intensities of bands were measured by densitometry using a CS Analyzer. KaiC showed a triplet band on SDS-PAGE (4, 9); the two upper bands correspond to phosphorylated KaiC (p-KaiC), and the lowest band corresponds to nonphosphorylated KaiC (np-KaiC). We calculated the relative amount of p-KaiC to total KaiC (the level of p-KaiC) from band intensities and analyzed the circadian oscillations of the level of p-KaiC using the program RAP (26).

In Vivo Bioluminescence Rhythm Assay

We measured the bioluminescence rhythms of the T. elongatus strains carrying a P_psbA1::Xl luxAB reporter gene at 41 °C using a newly developed bioluminescence monitoring apparatus with a robotic plate conveyor. The apparatus was 1.3 times as sensitive to luminescence as our previous apparatuses (27). We analyzed bioluminescence data by a modified cosiner method of RAP as previously described (26, 28). We examined the wild-type strain, a kaiB-null strain carrying a mutant kaiB gene with a nonsense mutation downstream of its initiation codon, and a strain carrying an additional mutant kaiB gene encoding KaiB_1–94, kaiB_1–94, in the kaiB-null genetic background (16, 29).

Assay for the Complex Formation of SasA with KaiC by Gel Filtration Chromatography

Reaction mixtures containing 1.5 μm SasA and 0.6 μm KaiC_DD were incubated in the presence or absence of 3.0 μm KaiB_WT or 6.0 μm KaiB_1–94 in HEPES reaction buffer containing 0.1 mm DTT at 25 °C for 18 h and analyzed by gel filtration chromatography on a Superdex 200/HR 10/300 column (GE Healthcare) equilibrated with HEPES reaction buffer containing 0.1 mm ATP, 5 mm MgCl₂, and 0.1 mm DTT at 4 °C. Aliquots of the chromatography fractions were subjected to SDS-PAGE on 15% gels, and the gels were stained with CBB. We determined the amount of SasA in each fraction by densitometry.

RESULTS

Oligomeric Structures of KaiB_1–94 and KaiB_WT in Solution

To determine the oligomeric structure of KaiB_1–94 in solution, we estimated the molecular mass of KaiB_1–94 by gel filtration chromatography in gel filtration buffer. KaiB_1–94 (subunit molecular mass, 10.9 kDa) eluted as a single peak (Fig. 1A) corresponding to a 23.4 ± 0.4-kDa protein (Fig. 1B and Table 1) that corresponds to the approximate molecular mass of a dimer, whereas KaiB_WT (subunit molecular mass, 12.4 kDa) eluted as a single peak (Fig. 1A) corresponding to a 55.4 ± 0.6-kDa protein (Fig. 1B and Table 1) that corresponds to the approximate molecular mass of a tetramer. We obtained similar results under varying buffer conditions (Table 1). In sedimentation equilibrium analysis, KaiB_1–94 sedimented as a dimer with a molecular mass of 23 kDa (Fig. 1C and Table 1), and KaiB_WT sedimented as a tetramer with a molecular mass of 48 kDa (Fig. 1D and Table 1), confirming the gel filtration chromatography results. Hereafter, when their concentrations are expressed, KaiB_WT is considered a tetramer and KaiB_1–94 a dimer.

FIGURE 1.

Determination of the oligomeric state of KaiB_1–94 and KaiB_WT. A, elution profiles of KaiB_1–94 and KaiB_WT by gel filtration chromatography are shown. KaiB_1–94 and KaiB_WT (the control) were subjected to analytical gel filtration chromatography at 4 °C on a Superdex75 5/150GL column equilibrated with 20 mm Tris-HCl buffer (pH 7.5) containing 150 mm NaCl. The elution positions of the molecular mass standards, conalbumin (75 kDa), carbonic anhydrase (29 kDa), ribonuclease (14 kDa), and aprotinin (6.5 kDa) are indicated above the elution profiles. Typical elution profiles from three independent experiments are shown. Black line, KaiB_1–94; gray line, KaiB_WT. mAu, milliabsorbance units. B, molecular masses of KaiB_1–94 and KaiB_WT were determined by gel filtration chromatography. Open circles, KaiB_1–94; gray circles, KaiB_WT; closed circles, the molecular mass standards. C and D, shown are sedimentation equilibrium analyses of KaiB_1–94 and KaiB_WT. The upper panels show the equilibrium profiles (○) displayed as the A₂₈₀ (Au) versus the radial distance and theoretical concentration profiles (solid line) for a single molecular species with a molecular mass of 22 and 48 kDa, respectively. The lower panels show the residual plots from the curve fitting. C, KaiB_1–94; D, KaiB_WT.

TABLE 1.

Molecular mass of KaiB_1–94

GF, gel filtration chromatography; SE, sedimentation equilibrium analysis; GFB, gel filtration buffer; SEB, sedimentation equilibrium buffer. Values for gel filtration chromatography are the means ± S.D. from triplicate measurements.

KaiB	Method	Buffer	Molecular mass (n = 3)
			kDa
KaiBWT	GF	GFB	55.4 ± 0.6
	GF	SEB	56.0 ± 1.0
KaiB1–94	GF	GFB	23.4 ± 0.4
	GF	SEB	22.1 ± 0.4
	GF	20 mm Tris-HCl (pH 7.5), 50 mm NaCl	22.5 ± 0.1
	GF	20 mm Tris-HCl (pH 7.5), 500 mm NaCl	25.6 ± 0
	GF	20 mm MES-NaOH (pH 6.5), 150 mm NaCl	24.0 ± 0.5
	GF	20 mm Tris-HCl (pH 8.5), 150 mm NaCl	22.5 ± 0.5
KaiBWT	SE	SEB	49.8
KaiB1–94	SE	SEB	23.0

Complex Formation of KaiB_1–94 and KaiB_WT with KaiC and the Stoichiometry of the KaiB_1–94-KaiC_DD and KaiB_WT-KaiC_DD Complexes

First, we examined the complex formation of KaiB_1–94 with KaiC_WT, KaiC_DD, and KaiC_AA by FCS analysis (supplemental Fig. S1). The diffusion time of Cy3-KaiB_1–94 incubated alone at 25 °C for 18 h was 0.13 ± 0.04 ms, and it increased in the presence of KaiC_DD (0.37 ± 0.09 ms). On the other hand, the diffusion time scarcely increased in the presence of KaiC_AA (0.16 ± 0.03 ms). These results indicate that KaiB_1–94 formed a complex with KaiC_DD, whereas it scarcely formed a complex with KaiC_AA. Furthermore, the diffusion time of Cy3-KaiB_1–94 increased in the presence of KaiC_WT when the phosphorylation level of KaiC_WT had been elevated to about 70% by KaiA (0.26 ± 0.03 ms), whereas it increased only slightly in the presence of KaiC_WT when the phosphorylation level of KaiC_WT (about 30%) had not been elevated by KaiA (0.18 ± 0.03 ms). Therefore, the complex formation of KaiB_1–94 with KaiC depends on the phosphorylation level of KaiC; the higher the phosphorylation level, the more efficient complex formation occurred. Probably, KaiB_1–94 associates with p-KaiC more strongly than with np-KaiC. These results are consistent with a previous observation that KaiB_WT associates with p-KaiC more strongly than with np-KaiC (30, 31). Hereafter, we used KaiC_DD to examine the complex formation of KaiB with KaiC because the phosphorylation level of KaiC_WT changes during incubation, especially in the presence of KaiB and/or KaiA (Ref. 4 and see Fig. 3A).

FIGURE 3.

Circadian oscillations in vitro and in vivo. A, shown are circadian oscillations in vitro. Generation of temperature-compensated circadian oscillations in vitro in the level of p-KaiC by KaiB_1–94 in an in vitro KaiABC clock system is shown. Reaction mixtures containing KaiA, KaiB_WT or KaiB_1–94, and KaiC_WT (0.5 μm each) were incubated in 20 mm Tris-HCl buffer (pH 7.5) containing 1 mm ATP, 5 mm MgCl₂, and 150 mm NaCl at 25 °C or 40 °C for the periods indicated and were subjected to SDS-PAGE on 12.5% gels. The gels were stained with CBB, and the relative amounts of np-KaiC and p-KaiC were estimated by densitometry. Typical photographs from three independent experiments are shown. Circadian oscillations in the level of p-KaiC were analyzed by RAP, and the simulated curves are shown. Values shown are the means ± S.D. from triplicate measurements. KaiBs added: open red circles, KaiB_1–94 (40 °C); open blue circles, KaiB_WT (40 °C); closed red circles, KaiB_1–94 (25 °C); closed blue circles, KaiB_WT (25 °C). Closed arrows, p-KaiC; open arrows, np-KaiC. B, shown are circadian bioluminescence rhythms of a T. elongatus P_psbA1::Xl luxAB reporter strain carrying kaiB_WT or kaiB_1–94 in a kaiB-null genetic background. Bioluminescence rhythms of T. elongatus P_psbA1::Xl luxAB reporter strains carrying kaiB_WT (WT, blue, n = 10), kaiB_1–94 (KaiB_1–94, red, n = 13), and no additional kaiB gene (KaiB-null, green, n = 75) in a kaiB-null background (the kaiB gene had a nonsense codon just downstream of its initiation codon) were measured at 41 °C. Data points and error bars indicate the mean ± S.D. from n independent samples. cps, counts per s.

Next we examined the KaiB_1–94-KaiC_DD complex by gel filtration chromatography followed by SDS-PAGE. KaiB_1–94 and KaiC_DD each eluted as a single peak, KaiB_1–94 with an apparent molecular mass of 25 ± 2 kDa and KaiC_DD with an apparent molecular mass of 319 ± 7 kDa (n = 3) (Fig. 2A). When KaiC_DD (1 μm) was incubated with an excess of KaiB_1–94 (5 μm) at 40 °C for 18 h (n = 3), the reaction products eluted as two peaks, one with a molecular mass of 26 ± 1 kDa (KaiB_1–94) and the other with a molecular mass of 350 ± 20 kDa (Fig. 2A), suggesting the formation of a KaiB_1–94-KaiC_DD complex. We also confirmed the formation of a KaiB_WT-KaiC_DD complex (366 ± 20 kDa) by gel filtration chromatography (Fig. 2A). The apparent molecular masses of the KaiB_1–94-KaiC_DD and KaiB_WT-KaiC_DD complexes were slightly higher than that of the KaiC_DD hexamer (Fig. 2A).

To determine the stoichiometry of the KaiB_1–94-KaiC_DD complex, we collected it by gel filtration chromatography (Fig. 2A, 9.0–9.5 ml) and estimated the amount of KaiB_1–94 and KaiC_DD it contained by SDS-PAGE (Fig. 2B) followed by densitometry (Fig. 2, C and D). When KaiC_DD (1 μm) was incubated with an excess of KaiB_1–94 (5 μm) at 40 °C for 18 h, the fraction containing the complex contained 3.8 ± 0.3 subunits of KaiB_1–94 per molecule of hexameric KaiC_DD (Fig. 2E). When we applied the same procedure to the KaiB_WT-KaiC_DD complex, the stoichiometry showed that it contained 4.2 ± 0.4 subunits of KaiB_WT per molecule of hexameric KaiC_DD (Fig. 2E), indicating that the KaiB_1–94-KaiC_DD complex consisted of two molecules of KaiB_1–94 (a dimer) and one molecule of hexameric KaiC_DD, whereas the KaiB_WT-KaiC_DD complex consisted of one molecule of KaiB_WT (a tetramer) and one molecule of hexameric KaiC_DD.

We confirmed these results using FCS with Cy3-NHS fluorescent dye ester (Cy3)-labeled KaiB proteins (Cy3-KaiB_1–94 and Cy3-KaiB_WT). When 0.2 μm Cy3-KaiB_1–94 and 0.1 μm Cy3-KaiB_WT were incubated in the presence of 0, 0.025, 0.05, 0.15, or 0.3 μm KaiC_DD, the diffusion time varied directly with the KaiC_DD concentration (Fig. 2F), indicating complex formation between KaiC_DD and Cy3-KaiB_1–94 or Cy3-KaiB_WT.

The counts per particle of Cy3-KaiB_1–94 (0.2 μm) also varied with KaiC_DD concentration (Fig. 2G). The counts per particle values of Cy3-KaiB_1–94 in the presence of KaiC_DD at molar ratios greater than 0.5 were approximately twice those in its absence, which suggested that one molecule of the Cy3-KaiB_1–94-KaiC_DD complex contained two molecules of the Cy3-KaiB_1–94 dimer (Fig. 2G). In contrast, counts per particle did not significantly change when Cy3-KaiB_WT (0.1 μm) was incubated with an excess of KaiC_DD (0.3 μm), which suggests that one molecule of the KaiB_WT-KaiC_DD complex contained one molecule of the KaiB_WT tetramer (Fig. 2G).

Interaction of KaiB_1–94 Versus KaiB_WT with KaiC_DD

Surface plasmon resonance analysis showed that the association rate (k_a) of KaiC_DD was 8 times as high with KaiB_1–94 as with KaiB_WT, and its dissociation rate (k_d) was 0.6 times as high (Table 2). Thus, the binding affinity of KaiB_1–94 for KaiC_DD was 15 times the binding affinity of KaiB_WT. We also examined the binding affinities of KaiA and SasA, other known KaiC-binding proteins, and determined that KaiB_1–94 had the highest binding affinity in the group (Table 2).

TABLE 2.

Kinetic and steady state parameters for the interaction of KaiB_WT, KaiB_1–94, KaiA, and SasA with KaiC_DD obtained by surface plasmon resonance analysis

Values are the means ± S.D. from triplicate measurements.

Protein	ka	kd	KD
	m−1s−1 (×104)	s−1 (×10−3)	m (×10−8)
KaiBWT	1.3 ± 0.057	0.46 ± 0.058	3.5 ± 0.38
KaiB1–94	10.4 ± 0.61	0.26 ± 0.040	0.24 ± 0.029
KaiA	3.9 ± 1.0	2.5 ± 0.10	6.8 ± 1.4
SasA	7.7 ± 1.1	1.2 ± 0.066	1.5 ± 0.24

Circadian Oscillations in the Level of p-KaiC in an in Vitro KaiABC Clock System and Circadian Gene Expression Rhythms in T. elongatus Cells

When 0.5 μm KaiA, KaiB (KaiB_1–94 or KaiB_WT), KaiC, and ATP were incubated together at 25 or 40 °C in an in vitro KaiABC clock system (4), the level of p-KaiC oscillated in the presence of KaiB_1–94 and KaiB_WT (Fig. 3A). At 40 °C, the period length was 21.7 ± 0.9 h for the oscillations driven by KaiB_1–94 and 22.6 ± 0.2 h for those driven by KaiB_WT (Fig. 3A), which was consistent with the circadian oscillations observed in T. elongatus cells (24.4 ± 0.3 h) (Ref. 16 and Fig. 3B). Thus, KaiB_1–94, like KaiB_WT, can generate circadian oscillations in the in vitro KaiABC clock system. At 25 °C, the period length was 21.7 ± 1.3 h for the oscillations driven by KaiB_1–94 and 23.3 ± 0.5 h for those driven by KaiB_WT (Fig. 3A), indicating that the period lengths were temperature-compensated in our system.

Circadian rhythms were normal in the kaiB-null host cells carrying kaiB_WT but showed a greatly reduced amplitude in those carrying kaiB_1–94 (Fig. 3B).

Inhibition of SasA-KaiC_DD Complex Formation by KaiB_1–94 and KaiB_WT

To determine why cyanobacterial cells expressing KaiB_1–94 showed weakened circadian gene expression rhythms even though KaiB_1–94 is able to generate normal circadian oscillations in vitro, we examined the effects of KaiB on the SasA-mediated clock output pathway, that is, on the formation of the SasA-KaiC complex.

Gel filtration chromatography yielded only a single peak for both KaiC_DD and SasA corresponding to a 340 ± 20-kDa protein and a 140 ± 10-kDa protein, respectively. When reaction mixtures containing 1.5 μm SasA and 0.6 μm KaiC_DD were incubated in the absence or presence of 3 μm KaiB_WT or 6 μm KaiB_1–94 at 25 °C for 18 h, the elution profile of the sample without KaiB showed 3 peaks (Fig. 4A) that we analyzed by SDS-PAGE (Fig. 4B). Peak 1 (>670 kDa, the molecular mass of the largest standard used) corresponded to the SasA-KaiC_DD complex, peak 2 (440 ± 50 kDa) corresponded to the other form of the SasA-KaiC_DD complex, and peak 3 (130 ± 5 kDa) corresponded to SasA. The amount of SasA contained in each fraction estimated by SDS-PAGE followed by densitometry showed that 34 ± 10% (n = 3) of the total SasA was unbound (Fig. 4C).

FIGURE 4.

Inhibition of the complex formation of SasA with KaiC_DD by KaiB_1–94 and KaiB_WT. A, elution profiles of gel filtration chromatography are shown. Reaction mixtures containing 1.5 μm SasA and 0.6 μm KaiC_DD in HEPES reaction buffer containing 0.1 mm DTT were incubated in the presence or absence of 6.0 μm KaiB_1–94 or 3.0 μm KaiB_WT at 25 °C for 18 h and were analyzed by gel filtration chromatography. Other conditions were as described in the Fig. 2 legend. Typical plots from three independent experiments are shown. KaiBs added: black solid line, KaiB_1–94; gray solid line, KaiB_WT; black dotted line, none. mAu, milliabsorbance units. B, shown is SDS-PAGE. Aliquots of the fractions (elution volumes, 7–17 ml) were subjected to SDS-PAGE on 15% gels, and the gels were visualized by CBB staining. Open arrowheads indicate KaiC_DD, and closed arrowheads indicate SasA. C, shown is the amount of SasA. The amount of SasA in each fraction was estimated with SDS-PAGE (Fig. 4B) using densitometry. KaiBs added: closed circles and black dotted line, none; open circles and black solid line, KaiB_1–94; gray circles and gray solid line, KaiB_WT. D, shown are SasA-induced changes in the diffusion times of the Cy3-KaiB_1–94 molecule and the Cy3-KaiB_WT molecule in the presence of KaiC_DD. Reaction mixtures containing Cy3-KaiB (0.2 μm Cy3-KaiB_1–94 or 0.1 μm Cy3-KaiB_WT) and 0.1 μm KaiC_DD were incubated in the presence or absence of the indicated amounts of SasA in HEPES reaction buffer containing 0.1 mm DTT at 25 °C for 18 h, and then the diffusion time was measured. The diffusion times of Cy3-KaiB_1–94 and Cy3-KaiB_WT in the absence of KaiC_DD and SasA were measured as controls. Values shown are the means ± S.D. from quadruple measurements. Other conditions were the same as described in the Fig. 2F legend. Open circles, Cy3-KaiB_1–94; open triangles, Cy3-KaiB_1–94 without KaiC_DD and SasA; gray circles, Cy3-KaiB_WT; gray triangles, Cy3-KaiB_WT without KaiC_DD and SasA.

The elution profile of the sample with 6 μm KaiB_1–94 showed three peaks (Fig. 4A); peak 1 (360 ± 10 kDa) corresponded to KaiC_DD and the KaiB_1–94-KaiC_DD complex, peak 2 (140 ± 10 kDa) corresponded to SasA, and peak 3 (20 ± 0 kDa) corresponded to KaiB_1–94. The presence of KaiB_1–94 all but extinguished the SasA-KaiC_DD complex and greatly increased free SasA (Fig. 4A). The free SasA fractions contained 92 ± 10% (n = 3) of the total SasA added (Fig. 4, B and C), which indicated nearly complete inhibition of SasA-KaiC_DD complex formation by KaiB_1–94. In the presence of 3 μm KaiB_WT, the elution profile of the sample showed four peaks (Fig. 4A) corresponding to the SasA-KaiC_DD complex (>670 kDa), the other form of the SasA-KaiC_DD complex, KaiC_DD and KaiB_WT-KaiC_DD complex (420 ± 40 kDa), SasA (140 ± 10 kDa), and KaiB_WT (50 ± 0 kDa). The presence of KaiB_WT slightly reduced the SasA-KaiC_DD complex peak and slightly increased the free SasA peak; the free SasA fractions contained 43 ± 5% (n = 3) of the total SasA added (control 34 ± 10%) (Fig. 4C), which suggests that KaiB_WT inhibited formation of the SasA-KaiC_DD complex, albeit very slightly. Thus, KaiB_1–94 was a stronger inhibitor of SasA-KaiC_DD complex formation than KaiB_WT.

We also examined KaiB inhibition of SasA-KaiC_WT complex formation by FCS analysis with Cy3-SasA (supplemental Fig. S2). The diffusion time of Cy3-SasA (0.2 μm) incubated alone at 25 °C for 18 h was 0.14 ± 0.00 ms. On the other hand, when Cy3-SasA was incubated with 0.2 μm KaiC_WT, its diffusion time increased to 0.30 ± 0.06 ms, indicating formation of a Cy3-SasA-KaiC_WT complex. The addition of 2.0 μm KaiB_1–94 almost completely canceled this diffusion time increase (0.16 ± 0.03 ms), indicating that KaiB_1–94 inhibited SasA-KaiC_WT complex formation as well as SasA-KaiC_DD complex formation (see above). However, the addition of KaiB_WT did not cancel the diffusion time increase under the conditions examined (diffusion time: 0.35 ± 0.03 and 0.33 ± 0.01 ms in the presence of 1.0 μm KaiB_WT and 2.0 μm KaiB_WT, respectively) (supplemental Fig. S2).

We examined SasA inhibition of KaiB-KaiC_DD complex formation by FCS analysis with Cy3-KaiB_1–94 and Cy3-KaiB_WT (Fig. 4D). In the absence of KaiC_DD, the diffusion time was 0.13 ± 0.02 ms for 0.2 μm Cy3-KaiB_1–94 and 0.11 ± 0.03 ms for 0.1 μm Cy3-KaiB_WT. In the presence of 0.1 μm KaiC_DD, it increased to 0.28 ± 0.03 ms for Cy3-KaiB_1–94 and 0.25 ± 0.05 ms for Cy3-KaiB_WT (Fig. 4D), indicating formation of the Cy3-KaiB_1–94-KaiC_DD and Cy3-KaiB_WT-KaiC_DD complexes. The diffusion time of 0.2 μm Cy3-KaiB_1–94 was slightly shorter in the presence of 0.3 μm SasA (0.23 ± 0.02 ms) than in its absence (0.28 ± 0.03) (Fig. 4D), which indicates that SasA weakly inhibited formation of the KaiB_1–94-KaiC_DD complex. In the presence of KaiC_DD, on the other hand, SasA reduced the diffusion time of 0.1 μm Cy3-KaiB_WT from 0.25 ± 0.05 to 0.13 ± 0.01 ms, the level it was in the absence of KaiC_DD (0.11 ± 0.03 ms) (Fig. 4D), which indicates that SasA almost completely inhibited formation of the KaiB_WT-KaiC_DD complex. Thus, SasA inhibited KaiB_WT-KaiC_DD complex formation more strongly than KaiB_1–94-KaiC_DD complex formation.

DISCUSSION

That KaiB_1–94 is a dimer (Fig. 1 and Table 1) but interacts with KaiC_DD in much the same way as tetrameric KaiB_WT (Fig. 2) and can generate normal circadian oscillations in vitro (Fig. 3A) indicates that KaiB tetramericity is not required for the generation of circadian oscillations per se. The tetrameric structure of KaiB, however, and its negatively charged C-terminal region seem to play an important role in the generation of circadian gene expression rhythms in vivo because the circadian bioluminescence rhythms of cyanobacterial cells expressing KaiB_1–94 in a ΔkaiB genetic background are greatly weakened (Ref. 16 and Fig. 3B). We demonstrated that whereas KaiB_WT inhibits the formation of the SasA-KaiC complex slightly, KaiB_1–94 inhibits it strongly (Fig. 4 and supplemental Fig. S2), probably because of the high affinity of KaiB_1–94 for KaiC_DD (Table 2).

SasA is a KaiC binding histidine kinase in the SasA-RpaA two-component regulatory system (32, 33) and is involved in the main clock output pathway. Ultimately, the time information of the KaiABC clock oscillator is transmitted to the transcription apparatus and generates genome-wide circadian transcription rhythms in cyanobacteria. Disruption of the sasA gene greatly lowers the level of kaiBC expression and the amplitude of kaiA and kaiBC expression rhythms (32). KaiB_1–94 competitively inhibits the SasA-KaiC interaction more strongly than KaiB_WT does due to its higher affinity for KaiC (Table 2); it is likely, therefore, that KaiB_1–94 hinders the SasA-KaiC interaction that is necessary for the transmission of the time information from the KaiABC clock oscillator to the transcription apparatus through the SasA-mediated clock output pathway (32, 33). We hypothesize that the defective rhythm phenotypes of cyanobacterial cells expressing KaiB_1–94 in a ΔkaiB genetic background follow from a KaiB_1–94-induced defect in the SasA-KaiC interaction involved in the SasA-mediated clock output pathway.

KaiB (KaiB_1–94 and KaiB_WT) inhibited formation of the SasA-KaiC (KaiC_DD and KaiC_WT) complex (Fig. 4, A–C, and supplemental Fig. S2), and SasA inhibited formation of the KaiB (KaiB_1–94 and KaiB_WT)-KaiC_DD complex (Fig. 4D). It is unknown whether the two proteins compete for an identical binding site on KaiC because the binding sites of KaiB and SasA on KaiC are not determined. Previously, the N-terminal domain of SasA was suggested to be homologous to KaiB (32). However, comparison between the NMR structure of SasA N-terminal domain and the x-ray crystal structure of KaiB demonstrated that KaiB and SasA N-terminal domain have different folds (34). Therefore, it is not likely that SasA and KaiB bind to the same site on KaiC. We have demonstrated by surface plasmon resonance analysis and gel filtration chromatography that the SasA binding site of KaiC is located on its N-terminal domain (21), as suggested by NMR analysis (35). The SasA inhibition of KaiB_WT-KaiC_WT complex formation has been demonstrated by Native-PAGE using proteins derived from Synechococcus (36).

Our demonstration that two molecules of KaiB_1–94 bind to one molecule of KaiC_DD (Fig. 2) suggests that two molecules of KaiB_1–94 dimer bind to one molecule of a KaiC hexamer. Because one molecule of KaiB_WT binds to one molecule of KaiC_DD (Fig. 2), it is likely that upon interaction with KaiC, the KaiB molecule changes from a tetramer to a dimer. This is consistent with the cryo-electron microscopy analysis of the KaiB_WT-KaiC_WT complex that revealed the possibility of KaiB binding to KaiC as two dimers (37). Based on the crystal structure of KaiB, we have proposed that the interdimer interface of the KaiB tetrameric molecule could be destabilized by changes in pH or ionic strength and dissociate into two dimers (16).

On one surface of the KaiB tetramer, a PC, which is the active site, is covered with two NRs, and the PC may be exposed for ligand binding by NR movement (Fig. 5A) (16). In agreement with this model, KaiB_1–94, which lacks NRs and has a continuously exposed PC, is a more stable binding partner for KaiC_DD than KaiB_WT (Table 2). It is likely that PC exposure by NR movement may play an important role in the interaction of KaiB with KaiC (Fig. 5, A and B). An alternative model is that KaiB associates with KaiC using a surface, which is exposed by the deletion of NRs on KaiB or the dimerization of KaiB. There are two areas on KaiC that are highly negatively charged, one around the pore opening and inside the pore of KaiC N-terminal domain and the other around the intersubunit interface of one of two adjacent KaiC N-terminal domains (Ref. 38 and supplemental Fig. S3). Possible electrostatic interaction between the PC on KaiB and the negatively charged surfaces of KaiC might occur.

FIGURE 5.

Model for the conformational change of KaiB. On one surface of the KaiB tetramer, a PC is covered with two NRs, and the active sites (16) and KaiA-interacting site (17) are located on the PC. Therefore, we hypothesize that the PC is exposed for ligand binding by NR movement (Refs. 16 and 39; A). The PC on KaiB_1–94, on the other hand, is always exposed, which allows it to interact with KaiC because it lacks the NRs and exists as a dimer (B). KaiB_1–94 is more stable than KaiB_WT as a binding partner for KaiC, which suggests that the KaiB binding site is located on the PC and that the PC may be exposed to KaiC by NR movement (A). Thus, KaiB_1–94 competitively inhibits the interaction of SasA with KaiC by its strong interaction with KaiC, which is necessary for the generation of normal circadian gene expression rhythms in cyanobacterial cells (C). On interaction with KaiC, the KaiB tetramer molecule may dissociate into two dimeric molecules that bind to one hexameric KaiC molecule (D). The tetrameric structure and NRs of the KaiB molecule are necessary for normal circadian gene expression rhythms in cells (D) but not for circadian oscillations in the in vitro KaiABC clock system.

Our demonstration that the stable interaction of KaiB_1–94 with KaiC inhibited the interaction of SasA with KaiC (Fig. 4) indicated that the C-terminal region of KaiB is necessary for driving normal circadian transcription rhythms through regulation of the KaiC-SasA interaction (Fig. 5, C and D). Furthermore, our recent observations are consistent with our model as follows. Electron spin resonance analysis demonstrated that the mobility of the spin labels introduced into the amino acid residues located on the PC greatly increased on incubation at 40 °C (39), which indicates that the local structure surrounding the spin labels on the PC is relaxed during incubation (39); the 64th residue located inside the KaiB molecule near the PC interacts with KaiA (17).

In the KaiABC clock oscillator, KaiA associates preferentially with np-KaiC (40) (KaiC_AA mimics np-KaiC) to elevate the phosphorylation level of KaiC (11–13). Then, KaiA dissociates from the p-KaiC (KaiC_DD mimics completely phosphorylated KaiC) (40). In contrast, KaiB associates preferentially with the p-KaiC (Ref. 6 and supplemental Fig. S1) and then reduces the phosphorylation level of KaiC (12, 14). A series of these reactions and interactions among Kai proteins generate circadian oscillations such as oscillations in the phosphorylation level (4) and ATPase activity (5) of KaiC and complex formation among Kai proteins (6, 7). SasA also associates preferentially with p-KaiC, which enhances the autophosphorylation of SasA (21). KaiB and SasA compete each other for KaiC (Fig. 4 and supplemental Fig. S2). Therefore, it is likely that KaiB regulates the normal SasA-KaiC interaction required for the transmission of time information from KaiC to the transcription apparatus, resulting in genome-wide transcription cycles in cyanobacterial cells (21, 32, 33). SasA trimer and KaiC hexamer associate each other at a 1:1 molar ratio at their N-terminal domains, and the phosphorylation states of their C-terminal domains affect the affinity of the interaction (21). Interestingly, the phosphorylation state of KaiC also affects the rigidity of the C-terminal domains of KaiC hexamer and the interaction between the N-terminal domains and C-terminal domains of the KaiC, and KaiC associates with KaiB when the structure of KaiC C-terminal domains is rigid (35).

KaiB and KaiC occur in Archaea and Proteobacteria as well as in cyanobacteria (41). The C-terminal cluster of negatively charged residues is highly conserved in cyanobacterial clock KaiBs (Ref. 16 and Fig. 6) but not in cyanobacterial non-clock KaiBs (such as Synechocystis KaiB2 (41)) and not in KaiB homologues in Archaea and Proteobacteria (Fig. 6). The negatively charged C terminus of KaiB likely plays an important role in the SasA-mediated clock output pathway through KaiB-KaiC interactions affecting the SasA-KaiC interaction. SasA is present only in cyanobacteria, which suggests that the negatively charged C-terminal region of cyanobacterial KaiB may have co-evolved with SasA as a component of the cyanobacterial circadian system.

FIGURE 6.

Amino acid sequence alignment of KaiB. We aligned the amino acid sequences of KaiB from three strains of cyanobacteria, two strains of archaea, and two strains of green-sulfur bacteria using ClustalX (42). The negatively charged residues (glutamate and aspartate are shaded in purple). The C-terminal deletion in KaiB_1–94 is indicated by underlining. Cyanobacterial strains: Synechococcus sp. strain PCC 7942; T. elongatus BP-1; Anabaena sp. strain PCC 7120; Synechocystis sp. PCC 6803 KaiB1; Synechocystis sp. PCC 6803 KaiB2. Archaeal strain: Methanosarcina mazei Goe1. Proteobacterial strains: Rhodospirillum rubrum ATCC 11170; Rhodopseudomonas palustris CGA009.