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Genes to Cells (2008) 13, 387-395. doi:10.1111/j.1365-2443.2008.01174.x
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ATPase activity and its temperature compensation of the cyanobacterial clock protein KaiC

 Reiko Murakami{dagger}, Ayumi Miyake{dagger}, Ryo Iwase, Fumio Hayashi, Tatsuya Uzumaki and Masahiro Ishiura*

Center for Gene Research, Nagoya University, Furocho, Chikusa, Nagoya 464-8602, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussions
 Experimental procedures
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussions
 Experimental procedures
 References
 
The circadian clock is an endogenous biological mechanism that generates autonomous daily cycles of physiological activities. Circadian rhythms are observed ubiquitously in prokaryotes and eukaryotes, and have the following characteristics (Dunlap 1999; Kondo & Ishiura 1999, 2000): (i) they persist under constant conditions, (ii) the phase is reset by external cues such as light/dark and low/high temperature signals, and (iii) the period shows temperature compensation.

Cyanobacteria are the simplest organisms known to exhibit circadian rhythms (Sweeney & Borgese 1989). Mutational analysis indicated that the cyanobacterial system is regulated by three clock genes—kaiA, kaiB and kaiC (Ishiura et al. 1998). KaiA is a homodimer of known crystal structure (Uzumaki et al. 2004). Its subunit is composed of three functional domains—the N-terminal amplitude-amplifier domain, the central period-adjuster domain and the C-terminal clock-oscillator domain (Uzumaki et al. 2004). The latter domain is responsible for the binding of KaiC, the enhancing activity of KaiC phosphorylation (Iwasaki et al. 2002; Williams et al. 2002; Uzumaki et al. 2004) and the generation of circadian oscillation (Uzumaki et al. 2004). KaiB has an unusual homotetramer structure comprising two asymmetrical dimers, as revealed by crystal structure (Hitomi et al. 2005; Iwase et al. 2005). KaiB is believed to attenuate the enhancing effect of KaiA on KaiC phosphorylation (Williams et al. 2002; Kitayama et al. 2003). KaiC is a monomer in the absence of ATP, which is oligomerized into a homohexamer by the addition of ATP (Mori et al. 2002; Hayashi et al. 2003), which binds preferentially to a high-affinity ATP-binding site in the N-terminal domain (Hayashi et al. 2004b). The KaiC hexamer has a hexagonal, pot-shaped structure composed of six identical dumbbell-shaped subunits as revealed by cryo-electron microscopy (Hayashi et al. 2003) and X-ray crystallography (Pattanayek et al. 2004). KaiC interacts with KaiA at its C-terminal domain (Hayashi et al. 2004b, 2006; Pattanayek et al. 2006). KaiC phosphorylates an adjacent subunit (Hayashi et al. 2006) and self dephosphorylates (Iwasaki et al. 2002; Williams et al. 2002; Hayashi et al. 2004a). The level of KaiC phosphorylation oscillates in cyanobacteria cells (Iwasaki et al. 2002), and the self-sustainable oscillation of KaiC phosphorylation has been reconstituted in vitro by incubating KaiC with KaiA and KaiB in the presence of ATP (Nakajima et al. 2005).

The KaiC subunit, based on protein phylogeny, has a duplicative structure of a RecA–DnaB family protein, and each half of the subunit (the N-terminal domain and the C-terminal domain) has a set of ATPase motifs (Walker motifs A and B, and a pair of deduced catalytic carboxylate Glu residues (CatEs) involved in ATP binding) (Ishiura et al. 1998; Leipe et al. 2000; Nishiwaki et al. 2000; Cox 2003; Hayashi et al. 2004b). The two domains show 23% similarity and 13% identity (Ishiura et al. 1998; Hayashi et al. 2004b). Various mutations in the two sets of ATPase motifs of KaiC cause arrhythmicity (Hayashi et al. 2004b), indicating that the motifs are critical to clock oscillation. The C-terminal ATPase motifs are involved in the intersubunit phosphorylation of the hexamer (Hayashi et al. 2006) at Ser431 and Thr432 (Nishiwaki et al. 2004; Xu et al. 2004) (KaiCC and KaiCCatE1- have the phosphorylation activity), whereas the N-terminal ATPase motifs are involved in KaiC hexamerization (Hayashi et al. 2004b, 2006) (KaiCN and KaiCCatE2- lacks the phosphorylation activity (Hayashi et al. 2004b, 2006)).

Other RecA–DnaB family proteins have an affinity for ATP and hydrolyze it to ADP and inorganic phosphate (Pi), but they have no phosphorylation sites corresponding to the Ser431 and Thr432 residues of KaiC (Leipe et al. 2000). In this paper, we prepared the highly purified samples of Thermosynechococcus elongatus (Yamaoka et al. 1978) KaiA, wild-type KaiC and various mutant KaiCs, and we examined whether the N-terminal and C-terminal domains carrying each set of ATPase motifs hydrolyzed ATP by the ATPase reaction. We found that both domains had weak ATPase activity. Furthermore, we demonstrated that the activity showed temperature compensation in KaiC but not in KaiCS431A/T432A, a mutant that lacked the phosphorylation sites and showed a higher ATPase activity than wild-type KaiC. We hypothesized that phosphorylation of KaiC suppresses its ATPase activity and is involved in its temperature-compensation mechanism.


   Results
Top
 Abstract
 Introduction
 Results
Discussions
 Experimental procedures
 References
 
ATPase activity of KaiC

Figure 1(A) shows the TLC results of the assay of KaiC hydrolysis of [{alpha}-32P] ATP and [{gamma}-32P] ATP. We attributed the small amount of [{alpha}-32P] ADP and [{gamma}-32P] Pi detected even in the absence of KaiC to the background levels and to the spontaneous breakdown of the ATPs during incubation. The amount of [{alpha}-32P] ADP and [{gamma}-32P] Pi released increased in the presence of KaiC, indicating that KaiC catalyzed the ATP hydrolysis.


Figure 1
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  		Figure 1  ATP hydrolysis by KaiC. (A) ATP hydrolysis by KaiC detected by TLC. Left lanes show the hydrolysis of [{gamma}-32P] ATP and [{alpha}-32P] ATP in the presence of KaiC or buffer. Right lanes show the separation of ATP, ADP, Pi and AMP, respectively. The black and white arrows indicate Pi and ADP, respectively. Radioactivity was detected by radio-TLC scanning by STORM 820 (GE Healthcare). KaiC (0.5 pmol hexamer/µL) was incubated with 1 mM ATP (0.2 µCi/µL [{gamma}-32P] ATP or 0.2 µCi/µL [{alpha}-32P] ATP) in 20 mM Tris–HCl buffer (pH 7.5) containing 5 mM MgCl2, 2 mM DTT and 150 mM NaCl at 25 °C for l h. Other conditions were the same as described in Experimental procedures. (B) Time course of ATP hydrolysis by KaiC in the presence or absence of KaiA. KaiC (0.5 pmol hexamer/µL) was incubated in 20 mM Tris–HCl buffer (pH 7.5) containing 1 mM ATP (0.2 µCi/µL [{gamma}-32P] ATP) at 25 °C for the periods indicated in the presence (closed circle) or absence (open circle) of KaiA (3.0 pmol dimer/µL). Other conditions were the same as described in the Fig. 1A legend. Means ± SDs were calculated from three independent measurements. (C) The relationship between the amount of ATP hydrolyzed (closed circle) and the amount of KaiC (open circle) in the reaction mixture. Purified KaiC was separated by gel filtration chromatography on a Superdex 200 column (GE Healthcare). Eight 10-µL fractions were incubated with [{alpha}-32P] ATP at 25 °C for 1 h. Other conditions were the same as described in the Fig. 1A legend. The fractions were analyzed by SDS-polyacrylamide gel electrophoresis, and the gels were visualized by CBB-staining. The amount of KaiC in each fraction was calculated by densitometry.

 
We also quantified the [{alpha}-32P] ADP released from [{alpha}-32P] ATP and found that it increased linearly during 4 h of incubation (data not shown). We calculated the amount of ATP hydrolyzed per hour at 25 °C to be 13.3 ± 1.1 molecules per molecule KaiC hexamer (data not shown). We similarly measured the amount of the [{gamma}-32P] Pi released from [{gamma}-32P] ATP and also observed that the increase was apparently linear for 4 h (Fig. 1B). In that assay, the amount of ATP hydrolyzed per hour at 25 °C was 6.0 ± 1.8 molecules per molecule KaiC hexamer (Fig. 1B). The reason the value was smaller for [{gamma}-32P] ATP than for [{alpha}-32P] ATP is that KaiC hydrolyzes ATP by two pathways—phosphorylation–dephosphorylation pathway (Iwasaki et al. 2002; Williams et al. 2002; Hayashi et al. 2004a), (in the presence of ATP, KaiC self-phosphorylates to phosphorylated-KaiC (p-KaiC), releasing ADP, and then p-KaiC self dephosphorylates, releasing Pi) and ATPase pathway (ADP and Pi are directly released from ATP by the ATPase reaction). [{alpha}-32P] ADP is directly released by the phosphorylation reaction as well as the ATPase reaction. On the other hand, although [{gamma}-32P] Pi is directly released by the ATPase reaction, it is released by the phosphorylation reaction only when it is immediately followed by the dephosphorylation reaction. In our previous assays, the amount of the [{gamma}-32P] Pi from [{gamma}-32P] ATP incorporated into KaiC increased linearly for at least 2 h (Hayashi et al. 2006), and the phosphorylation level of KaiC also increased linearly for at least 1 or 2 h (Hayashi et al. 2004b). Thus, the difference is explained by the accumulation of phosphorylated KaiC (Iwasaki et al. 2002; Williams et al. 2002; Hayashi et al. 2004a) due to the slow dephosphorylation reaction, and the [{gamma}-32P] Pi released from [{gamma}-32P] ATP for 1- or 2-h reactions can reflect the ATPase activity of KaiC.

The amount of ATP hydrolyzed per hour by one molecule of KaiC hexamer (ATPase activity) was small. To exclude the possibility that the KaiC preparations we used were contaminated with other proteins involved in ATP hydrolysis, we examined the relationship between the amount of ATP hydrolyzed and the amount of KaiC by gel filtration chromatography of KaiC preparations purified by ion-exchange chromatography as described in Experimental procedures. The amount of [{alpha}-32P] ADP released from [{alpha}-32P] ATP by fractions corresponded well to the amount of KaiC in the fractions as determined by densitometry, especially in peak fractions 4 and 5 (Fig. 1C), indicating that KaiC had ATP hydrolysis activity. We confirmed this by showing that KaiCs carrying mutations in the catalytic Glu residues of the ATPase motifs (KaiCN CatE1- and KaiCC CatE2-) had no significantly detectable ATP hydrolysis activity (Fig. 2B).


Figure 2
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  		Figure 2  ATPase activities of KaiCN and KaiCC. (A) Time course of ATP hydrolysis by KaiCN (red triangle) and KaiCC (blue circles). KaiCs (0.5 pmol hexamer/µL) were incubated with 1 mM ATP (0.2 µCi/µL [{gamma}-32P] ATP) at 25 °C for the periods indicated in the presence (closed, solid line) or absence (open, dashed line) of KaiA (3.0 pmol/µL). Other conditions were the same as described in the Fig. 1A legend. Means ± SDs were calculated from three independent measurements. (B) Effects of CatEs mutations in KaiC on ATP hydrolysis. KaiCs (0.5 pmol/µL) were incubated with 1 mM ATP (0.2 µCi/µL [{gamma}-32P] ATP) at 25 °C for 2 h. Other conditions were the same as described in the Fig. 2A legend. Means ± SDs were calculated from three measurements. WT, wild-type.

 
ATPase activity of the KaiC N-terminal domain

To examine the ATPase activity of the KaiC N-terminal domain, we used the N-terminal domain protein, KaiCN (residues 1–251), which has a set of ATPase motifs but no phosphorylation sites and no phosphorylation activity (Hayashi et al. 2006). We detected ATP hydrolysis using [{gamma}-32P] ATP (0.70 ± 0.30 molecules ATP per hour per molecule of KaiCN hexamer at 25 °C) (Fig. 2A). Its activity was about 20% that of full-length KaiC (Fig. 2B). The activity of KaiCN CatE1-, in contrast, was only 13% of that of KaiCN (Fig. 2B), confirming that the N-terminal ATPase motifs were responsible for the ATPase activity of KaiCN.

ATPase activity of KaiC C-terminal domain

We examined the ATP hydrolysis of KaiC C-terminal domain protein, KaiCC (residues 252–518), which has a set of ATPase motifs and intersubunit phosphorylation activity (Hayashi et al. 2006). The ATP hydrolysis activity of KaiCC was 0.4 ± 0.2 molecules ATP per hour per molecule of KaiCC hexamer at 25 °C) (Fig. 2A). Its activity was only 6% of that of full-length KaiC and about 30% of that of KaiCN (Fig. 2B). KaiCC CatE2-, in contrast, showed no detectable ATP hydrolysis activity (Fig. 2B), confirming that the KaiCC ATPase motifs were responsible for the ATP hydrolysis by KaiCC.

The total activities of an equimolar mixture of KaiCN and KaiCC was about 30% of that of full-length KaiC, or 1.6 ± 0.1 molecules ATP per hour per each molecule (as hexamer) of KaiCN and KaiCC (data not shown), suggesting that the direct connection between the C- and N-terminal domains of KaiC is required for the full expression of ATP hydrolysis activity.

To distinguish the ATPase and phosphorylation–dephosphorylation pathways of KaiCC, we examined the ATPase activity of KaiCC S431A/T432A using [{gamma}-32P] ATP (Fig. 3A). We found that KaiCC S431A/T432A hydrolyzed ATP, indicating that the KaiC C-terminal domain had ATPase activity.


Figure 3
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  		Figure 3  Effects of the S431A and T432A mutations on KaiC ATPase activity. (A) Effects of the S431A and T432A mutations on the ATPase activities of KaiC, KaiCCatE2- and KaiCC. Wild-type (WT) and mutant KaiCs (0.5 pmol hexamer/µL) were separately incubated with 1 mM ATP (0.2 µCi/µL [{gamma}-32P] ATP) at 25 °C for 2 h in the presence (white bar) or the absence (black bar) of KaiA (3.0 pmol/µL). Other conditions were the same as described in the Fig. 2A legend. Means ± SDs were calculated from five independent measurements. The P-values were calculated by Student's t-test and are indicated by * (< 0.05) or ** (< 0.1). (B) Temperature compensation of the ATPase activity of KaiC and mutant KaiCs. KaiCs were incubated for 2 h at 25 °C (black bar), 37 °C (gray bar) or 50 °C (white bar). Other conditions were the same as described in the Fig. 3A legend. Means ± SDs were calculated from five independent measurements.

 
Effect of phosphorylation on KaiC ATPase activity

Surprisingly, ATPase activity was higher for KaiCS431A/T432A (11.3 ± 1.9 molecules ATP per hour per hexamer) and KaiCC S431A/T432A (4.7 ± 0.1 molecules ATP per hour per hexamer) than for KaiC and KaiCC (Fig. 3A), indicating that the lack of phosphorylation enhanced ATPase activity and suggesting that KaiC phosphorylation suppresses the ATPase activity of KaiC and KaiCC.

ATPase activity of the KaiC N-terminal domain, on the other hand, did not differ significantly for KaiCCatE2- (2.5 ± 0.4 molecules ATP per hour per molecule hexamer) and KaiCCatE2- S431A/T432A (2.4 ± 0.5 molecules ATP per hour per molecule hexamer) (Fig. 3A), indicating that the activity was not affected by mutations at the KaiC phosphorylation sites. The ATPase activities of full-length KaiC and KaiCC were increased by the S431A and T432A mutations about 350% and 270%, respectively (Fig. 3A).

Effect of KaiA on the ATPase activity of KaiC

The addition of KaiA enhanced KaiC hydrolysis of [{gamma}-32P] ATP about 60% (Fig. 1B). Because KaiA enhances the phosphorylation of KaiC (Williams et al. 2002; Iwasaki et al. 2002; Uzumaki et al. 2004), we examined the effect of KaiA on the ATPase activity of KaiC, using KaiCS431A/T432A and KaiCC S431A/T432A mutants lacking the phosphorylation sites. In fact, KaiA enhanced the ATPase activities of KaiCS431A/T432A about 50% and that of KaiCC S431A/T432A about 30% (Fig. 3A), indicating that KaiA enhanced the ATPase activity of both KaiC and KaiCC. KaiA had no effect, however, on the hydrolysis of [{gamma}-32P] ATP by KaiCN, which is consistent with our previous observations that KaiCN does not interact with KaiA (Hayashi et al. 2006).

The ATPase activity of KaiCCatE2- was 2.5 ± 0.2 molecules ATP per hour per molecule KaiC hexamer at 25 °C (Fig. 3A), and that of KaiCN was 1.8 ± 0.2 molecules ATP per hour per molecule of KaiCN hexamer at 25 °C (Fig. 2B). KaiA enhanced the activity only of KaiCCatE2- about 30% (Figs 2A and 3A), suggesting that KaiA binding to full-length KaiC at the C-terminal domain enhances the ATPase activity of the N-terminal domain. KaiA also enhanced the ATPase activity of KaiCCatE2- S431A/T432A (Fig. 3A), suggesting that KaiA enhancement of the KaiC N-terminal domain was independent of KaiC C-terminal phosphorylation.

In the time-course experiment, KaiA enhanced ATP hydrolysis by KaiCC at 1 h about 320% (Fig. 2A, 1 h). The interaction of KaiA with KaiCC decreased the Km value of KaiCC ATPase about 50% and increased its Vmax value about 230% (Table 1). These results are consistent with our previous observations that KaiA interacts with KaiC at the C-terminal domain (Hayashi et al. 2006).


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  		Table 1  Kinetics parameters of KaiC ATPase
 
Temperature compensation of KaiC ATPase activity

ATPase activity was temperature compensated in KaiC (Fig. 3B). The Q10 values between 25 °C and 50 °C were 0.97 ± 0.05 in the absence of KaiA and 1.01 ± 0.03 in its presence. In KaiCS431AT/432A, however, ATPase activity varied directly with the temperature (Fig. 3B), and the Q10 values were 1.54 ± 0.23 in the absence of KaiA and 1.83 ± 0.16 in its presence. Thus, KaiC phosphorylation was involved in the temperature compensation of its ATPase activity.


   Discussions
Top
 Abstract
 Introduction
 Results
 Discussions
Experimental procedures
 References
 
The rate (6.0 ± 1.8 molecules ATP per hour per molecule KaiC hexamer at 25 °C) was extremely low compared to the rates reported for other RecA–DnaB family proteins. For example, RecA, an ATPase involved in the DNA repair process and RecA function, requires the formation of an active RecA–DNA filament comprising multiple RecA, ATP and DNA components (Cox 2003). Brenner et al. estimated the ATP hydrolysis rate of RecA in the presence of DNA as 30 molecules of ATP per minute per monomer (Brenner et al. 1987), or about 1800 times the KaiC rate. This very weak ATPase activity of KaiC is interesting because the apparent rate of the circadian clock is extremely low, about one cycle per 24 h.

We demonstrated that both the N- and C-terminal domains of KaiC have weak ATPase activity (Fig. 2). The ATPase activity of the N-terminal domain (KaiCN) was about two times that of the C-terminal domain (KaiCC) (Fig. 2), and the Km for KaiCC ATPase was about 10 times the Km for KaiCN ATPase (Table 1). This observation is consistent with our previous report that ATP preferentially binds to the N-terminal high-affinity ATP-binding site of KaiC (Hayashi et al. 2004b). The C-terminal ATPase motifs of KaiC are involved in KaiC phosphorylation activity (Hayashi et al. 2004b, 2006), and as demonstrated here, KaiC C-terminal ATPase motifs are also involved in the ATPase activity of KaiC C-terminal domain (Fig. 2). Because other RecA–DnaB family proteins have no phosphorylation sites corresponding to the C-terminal Ser431 and Thr432 residues of KaiC (Leipe et al. 2000), the phosphorylation, which regulates ATPase activity, is a unique feature of the KaiC C-terminal domain.

One characteristic feature of the circadian clock is that the period length is kept almost constant under varying conditions (Dunlap 1999; Kondo & Ishiura 1999, 2000; Onai et al. 2004). We demonstrated here that ATPase activity was temperature-compensated in wild-type KaiC but not in KaiCS431A/T432A, a mutant that lacks KaiC phosphorylation sites (Fig. 3B).

We also demonstrated that ATPase activity in the KaiC C-terminal domain was enhanced in the KaiCS431A/T432A (Figs 3 and 4). How KaiC phosphorylation suppresses the ATPase activity of KaiC C-terminal domain has several possible explanations: (i) simple competition between the ATPase and phosphorylation reactions of KaiC results in suppression of the ATPase activity; (ii) the phosphates at the KaiC phosphorylation sites inhibit the binding of ATP to the KaiC C-terminal low-affinity ATP-binding sites (C-terminal ATPase motifs), either because the C-terminal Ser431 and Thr432 residues of KaiC lie within 10 Å of the {gamma}-phosphates of ATP (as revealed by the crystal structure of KaiC (Pattanayek et al. 2004)) or because of the negative charges of the phosphates; and (iii) phosphorylation induces a conformational change that suppresses the ATPase activity. The S431A and T432A mutations partially suppress the oligomerization of KaiCC (Hayashi et al. 2006).


Figure 4
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  		Figure 4  Model of KaiC hexamer. Each dumbbell along the vertical axis corresponds to a KaiC subunit. The upper and lower spheres of the dumbbell correspond to the N-terminal and C-terminal domains, respectively. The KaiC subunit has a duplicated structure, and each half has a set of ATPase motifs (blue circles). The N-terminal domain has its own ATPase activity, whereas the C-terminal ATPase motifs are involved in the intersubunit phosphorylation at Ser431 and Thr432 residues (green circle) as well as in the ATPase activity of C-terminal domain. The phosphorylation of KaiC suppresses ATPase activity in the C-terminal domain. KaiA binds directly to the KaiC C-terminal domain and enhances both the ATPase activity and intersubunit phosphorylation of the KaiC C-terminal domain. KaiA bound to KaiC via the KaiC C-terminal domain also indirectly enhances the ATPase activity of KaiC N-terminal domain.

 
We conclude 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 hypothesize the following temperature-compensation mechanism: (i) The C-terminal phosphorylation sites of a KaiC subunit in the KaiC hexamer are phosphorylated by the C-terminal domain of an adjacent KaiC subunit; (ii) the phosphorylation suppresses the ATPase activity of the KaiC C-terminal domain; (iii) the phosphorylated KaiC spontaneously dephosphorylates, resulting in the recovery of ATPase activity; and (iv) the phosphorylation of KaiC is involved in the temperature-compensation mechanism of KaiC ATPase.

Based on our hypothesis, the ATPase activity of KaiC should be initially high and then should become gradually lower by increasing its phosphorylation level. KaiC monomer purified from Escherichia coli in the absence of ATP was already partially phosphorylated (about 30% of KaiC was phosphorylated) (Hayashi et al. 2004b; Uzumaki et al. 2004). When KaiC incubated with ATP, KaiC was hexamerized, and its phosphorylation level increased (to about 40% or 60% in the absence or presence of KaiA, respectively, during an initial 2-h incubation) (Hayashi et al. 2004b). One of the reason why the rate of ATP hydrolysis by KaiC became lower at 8-h of incubation (Fig. 1B) would be the increased suppression of the ATPase activity by the increase of KaiC phosphorylation level.

We believe that the N-terminal ATPase motifs of KaiC are mainly responsible for its hexamerization (Hayashi et al. 2004b). ADP cannot induce that hexamerization (Hayashi et al. 2003), so the ATP hydrolysis at the KaiC N-terminal domain may destabilize KaiC's hexamer structure. It is possible that the ATP hydrolysis by the KaiC N-terminal domain may play a critical role in the shuffling of KaiC subunits among its hexamers (Kageyama et al. 2006). It is also possible that reversely, the oligomerization state of KaiC (monomer or hexamer) may affect KaiC ATPase activity (N-terminal and C-terminal ATPase activities) and then the ATP-binding state of KaiC.

KaiA enhanced all the ATPase activities of the KaiCs we examined (Figs 2–4). KaiA also accelerates the phosphorylation of KaiC (Iwasaki et al. 2002; Williams et al. 2002; Uzumaki et al. 2004). Thus, KaiA enhances both the ATPase and phosphorylation activities of the KaiC C-terminal domain.

When KaiA interacted with the KaiC C-terminal domain, it enhanced directly the ATPase activity of the C-terminal domain (Fig. 2A) and it also enhanced indirectly that of the N-terminal domain (Figs 2A, 3A and 4), probably because it could not interact directly with the N-terminal domain (Hayashi et al. 2006). We cannot completely exclude the possibility, however, that the N-terminal domain interacts weakly with KaiA because of a possible interaction between KaiA and the KaiC N-terminal domain as shown in the yeast two-hybrid system (Taniguchi et al. 2001).

The enhancement of KaiC ATPase activity by KaiA is likely to be independent on its suppression by KaiC phosphorylation, because (i) KaiA enhanced ATPase activity in KaiC and KaiCC as much as in KaiCS431A/T432A and KaiCC S431A/T432A (Figs 2A and 3A); and (ii) KaiA, but not the S431A and T432A mutations in KaiC, enhanced ATPase activity in KaiCCatE2- (Fig. 3A).

Very recently, Terauchi et al. estimated the ATP hydrolysis activities (the sum of ATPase and phosphorylation activities) of KaiC derived from Synechococcus sp. strain PCC7942 (Synechococcus KaiC) and Synechococcus KaiCS431A/T432A by monitoring ADP production using an HPLC with a titanium dioxide column (Terauchi et al. 2007). Terauchi et al. reported that both Synechococcus KaiC and KaiCS431A/T432A showed temperature compensation in their ATP hydrolysis activities in a temperature range of 25–35 °C (Terauchi et al. 2007). Although our observed difference in the ATPase activity of T. elongatus KaiCS431A/T432A between at 25 °C and 37 °C was small (about 120%), examinations of a wider temperature range of 25–50 °C could reveal a significant difference in its ATPase activity (Fig. 3B).


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussions
 Experimental procedures
References
 
Preparation of KaiA, wild-type KaiC and mutant KaiCs

The plasmids that express KaiA and wild-type KaiC derived from T. elongatus were described previously (Hayashi et al. 2003, 2004a). Plasmids expressing mutant KaiCs—KaiCN (an N-terminal domain protein of KaiC, residues 1–251), KaiCC (a C-terminal domain protein of KaiC, residues 252–518), KaiCCatE1- (a mutant KaiC carrying a Gln substitution at each of the two N-terminal catalytic Glu residues, E78 and E79, CatE1), KaiCCatE2- (a mutant KaiC carrying a Gln substitution at each of the two C-terminal catalytic Glu residues, E318 and E319, CatE2) and KaiCC S431A/T432A (a mutant KaiCC carrying the S431A and T432A mutations)—were also described previously (Hayashi et al. 2004b, 2006). We constructed each of the following plasmids expressing mutant KaiCs by the in vitro mutagenesis method using the polymerase chain reaction (PCR) as described previously (Hayashi et al. 2004b)—KaiCS431A/T432A (a mutant KaiC carrying an Ala substitution at two KaiC phosphorylation sites, Ser431 and Thr432 residues), KaiCC CatE2- (a mutant KaiCC carrying the two CatE2– mutations), and KaiCCatE2- S431A/T432A (a mutant KaiC carrying both the CatE2– and S431A/T432A mutations). We transferred the plasmids into E. coli BL21 and grew the cells in Luria–Bertani broth (LB) or Terrific broth (TB). We purified KaiA and KaiC, as well as the mutants, as described previously (Hayashi et al. 2003, 2004a). Briefly, glutathione-S-transferase (GST)-KaiA and GST-KaiCs were purified by affinity chromatography with Glutathione Sepharose 4B beads (GE Healthcare, Buckinghamshire, UK), and the GST-tags were removed by PreScission protease (GE Healthcare) digestion. KaiA and KaiCs were purified by ion-exchange chromatography on a Q-Sepharose column (GE Healthcare) and a MonoQ column (GE Healthcare) followed by gel filtration chromatography on a Superdex 200 column (GE Healthcare).

Protein analysis

Purified proteins were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 15% gels, and the gels were stained with Coomassie Brilliant Blue (CBB) (Laemmli 1970). We estimated protein concentration using the Bio-Rad Protein Assay (Bio-Rad, Philadelphia, PA).

Assays of ATPase activity

The monomers of KaiCs were incubated with 1 mM ATP (0.2 µCi/µL [{gamma}-32P] ATP or [{alpha}-32P] ATP) in 20 mM Tris–HCl buffer (pH 7.5) containing 5 mM MgCl2, 2 mM DTT, and 150 mM NaCl at 25 °C, 37 °C or 50 °C for various periods of time in the presence or absence of KaiA. After the reaction, we separated the nucleotides by thin layer chromatography (TLC) with 1 M formic acid and 0.4 M LiCl and PEI-Cellulose F (Sadis & Hightower 1992) (Merck, Frankfurter, Germany). We quantified radioactivity by radio-TLC scanning using STORM 820 (GE Healthcare), and expressed the results as the means ± SDs of triplicate or quintuplicate measurements upon subtraction of the buffer blanks.


   Acknowledgements
 
We thanks Satoko Ogawa and Kumiko Tanaka for technical support and Dr Miriam Bloom (SciWrite Biomedical Writing and Editing Services) for professional editing. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), ‘National Project on Protein Structural and Function analysis’ promoted by MEXT. The Division of Biological Science, Graduate School of Science, Nagoya University, was supported by a 21st Century Center of Excellence grant from MEXT.


   Footnotes
 
{dagger}These authors have equally contributed to this study. Back

Communicated by: Shunsuke Ishii

* Correspondence: Email: ishiura{at}gene.nagoya-u.ac.jp


   References
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Received: 7 November 2007
Accepted: 8 January 2008




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