The ATP-Mediated Regulation of KaiB-KaiC Interaction in the Cyanobacterial Circadian Clock

Risa Mutoh; Atsuhito Nishimura; So Yasui; Kiyoshi Onai; Masahiro Ishiura

doi:10.1371/journal.pone.0080200

Abstract

The cyanobacterial circadian clock oscillator is composed of three clock proteins—KaiA, KaiB, and KaiC, and interactions among the three Kai proteins generate clock oscillation in vitro. However, the regulation of these interactions remains to be solved. Here, we demonstrated that ATP regulates formation of the KaiB-KaiC complex. In the absence of ATP, KaiC was monomeric (KaiC^1mer) and formed a complex with KaiB. The addition of ATP plus Mg²⁺ (Mg-ATP), but not that of ATP only, to the KaiB-KaiC^1mer complex induced the hexamerization of KaiC and the concomitant release of KaiB from the KaiB-KaiC^1mer complex, indicating that Mg-ATP and KaiB compete each other for KaiC. In the presence of ATP and Mg²⁺ (Mg-ATP), KaiC became a homohexameric ATPase (KaiC^6mer) with bound Mg-ATP and formed a complex with KaiB, but KaiC hexamerized by unhydrolyzable substrates such as ATP and Mg-ATP analogs, did not. A KaiC N-terminal domain protein, but not its C-terminal one, formed a complex with KaiB, indicating that KaiC associates with KaiB via its N-terminal domain. A mutant KaiC^6mer lacking N-terminal ATPase activity did not form a complex with KaiB whereas a mutant lacking C-terminal ATPase activity did. Thus, the N-terminal domain of KaiC is responsible for formation of the KaiB-KaiC complex, and the hydrolysis of the ATP bound to N-terminal ATPase motifs on KaiC^6mer is required for formation of the KaiB-KaiC^6mer complex. KaiC^6mer that had been hexamerized with ADP plus aluminum fluoride, which are considered to mimic ADP-Pi state, formed a complex with KaiB, suggesting that KaiB is able to associate with KaiC^6mer with bound ADP-Pi.

Citation: Mutoh R, Nishimura A, Yasui S, Onai K, Ishiura M (2013) The ATP-Mediated Regulation of KaiB-KaiC Interaction in the Cyanobacterial Circadian Clock. PLoS ONE 8(11): e80200. https://doi.org/10.1371/journal.pone.0080200

Editor: David E. Somers, Ohio State University, United States of America

Received: March 14, 2013; Accepted: October 1, 2013; Published: November 11, 2013

Copyright: © 2013 Mutoh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by grants (KAKENHI) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) to MI and RM and the Sasakawa Scientific Research Grant from the Japan Science Society to AN. RM was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Circadian rhythms—oscillations that regulate metabolic and behavioral activity in approximately 24-h periods—are observed in almost all organisms from prokaryotes to eukaryotes. Cyanobacteria are the simplest organisms to exhibit circadian rhythms [1]. The gene cluster kaiABC that is essential for the generation of circadian rhythms has been cloned and analyzed in the cyanobacterium Synechococcus sp. strain PCC 7942 (hereafter Synechococcus) [2]. The cyanobacterial clock oscillator is composed of only three clock proteins, KaiA, KaiB, and KaiC. Circadian oscillations are generated at the phosphorylation level [3] and ATPase activity [4] of KaiC and the formation of complexes between the proteins [5,6]. KaiC is a homohexameric ATP-binding protein with autokinase activity [7-9], very weak temperature-independent (temperature-compensated) ATPase activity [4,10], and autophosphatase activity [11,12]. The KaiC subunit has a duplicated structure composed of N-terminal and C-terminal domains, and each has a series of ATPase motifs (a Walker's motif A, a Walker's motif B, and a catalytic glutamate (CatE; We named the N-terminal CatE and C-terminal CatE as CatE1 and CatE2, respectively.)) (Figure 1A) [2,13]. KaiC has two phosphorylation sites, Ser431 and Thr432, in its C-terminal domain (Figure 1A). A mutant KaiC with aspartate substitutions at the two phosphorylation sites, KaiC_S431D&T432D (KaiC_DD), is considered to mimic the fully phosphorylated state of KaiC [14], and a mutant KaiC with alanine substitutions at the two phosphorylation sites, KaiC_S431A&T432A (KaiC_AA), is considered to mimic fully unphosphorylated state of KaiC [15]. KaiC_DD showed higher affinity with KaiB than KaiC_AA [5,15-18]. KaiA is a homodimeric protein [9,19,20] that enhances the phosphorylation level [9,21,22] and ATPase activity [4,10] of KaiC. KaiB is a homotetrameric protein [23-25], and it has a positively charged cleft flanked by two negatively charged ridges [24]. KaiB suppresses the autokinase activity [22,26] and ATPase activity [4] of KaiC. KaiC associates with KaiA [8,9] and KaiB [25,27], and KaiB also associates directly with KaiA [16,28,29]. These interactions are essential for the generation of clock oscillation. On interaction with hexameric KaiC (KaiC^6mer), tetrameric KaiB (KaiB^4mer) likely dissociates into two dimeric KaiB (KaiB^2mer) and forms a complex comprising one molecule of KaiC^6mer and two of KaiB^2mer [17]. Previously, we have shown that KaiB_1-94-KaiC_DD^6mer complex (350 ± 20 kDa) consists of two molecules of KaiB_1-94 (dimeric) and one molecule of KaiC_DD^6mer and also have shown data suggesting that KaiB_WT-KaiC_DD^6mer complex (366 ± 20 kDa) consists of two molecules of dimerized KaiB_WT (KaiB_WT^2mer) and one molecule of KaiC_DD^6mer [17]. The histidine kinase SasA, which is the first component of the main output pathway that transduces clock oscillation to genome-wide transcription cycles [30], competes with KaiB to form a complex with KaiC [17], and SasA and KaiC associate through their N-terminal domains [14]. How the KaiB-KaiC interaction is regulated, however, remains unknown.

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Figure 1. The sequence motifs of KaiC and KaiB-KaiC complex formation.

A. A diagram of the sequence motifs of KaiC. B. Time courses of KaiB-KaiC_DD^6mer complex formation. KaiB_WT (1 μM) and KaiB_1-94 (2 μM) were separately incubated with 1 μM KaiC_DD^6mer in reaction buffer containing Mg-ATP at 4 °C or 40 °C for the various periods indicated, and then aliquots of the reaction mixtures were subjected to native PAGE on 10 % gels. After staining the gels with CBB to visualize proteins, we estimated the amounts of KaiBs-KaiC_DD^6mer complex by densitometry. We used the maximum value obtained at a time of 6 h as the maximum value. KaiBs added and temperature conditions: open circles, KaiB_WT (40 °C); closed circles, KaiB_1-94 (40 °C); open squares, KaiB_1-94 (4 °C). C. A native PAGE gel of the reaction products of KaiC_DD^1mer with KaiB_1-94. Reaction mixtures containing 15 μM KaiB_1-94 and 5 μM KaiC_DD^1mer in reaction buffer were incubated at 4 °C for 6 h and then subjected to native PAGE. Proteins were visualized by CBB staining of the gel. Because KaiB_1-94 has an isoelectric point of 9.7, it moved in the opposite direction and could not detect by native PAGE. D. A native PAGE gel of the reaction products of KaiC_DD^1mer with KaiB_WT. Reaction mixtures containing 7.5 μM KaiB_WT and 5 μM KaiC_DD^1mer in reaction buffer were incubated at 4 °C for 6 h and then subjected to native-PAGE. E. Time course of KaiB_1-94-KaiC_DD^1mer complex formation. Reaction mixtures containing 5 μM KaiB_1-94 and 5 μM KaiC_DD^1mer in reaction buffer were incubated at 4 °C for the periods indicated and then subjected to native PAGE. We used the value obtained at a time of 9 h as the maximum value. A typical experimental data is shown. F. KaiB_1-94 concentration-dependence of the KaiB_1-94-KaiC_DD^1mer complex formation. Reaction mixtures containing 1 μM KaiC_DD^1mer and various amounts (0.5, 0.75, 1.0, 1.25, 1.5, 2.0, 2.5, 3.0, and 4.0 μM) of KaiB_1-94 in reaction buffer were incubated at 4 °C for 6 h and then subjected to native PAGE. We used the value obtained at a KaiB_1-94 concentration of 4 μM as the maximum value. A typical experimental data is shown.

https://doi.org/10.1371/journal.pone.0080200.g001

Here, we investigated the interaction of KaiC with KaiB in vitro and found that KaiC associates with KaiB via its N-terminal domain and that ATP regulates the KaiB-KaiC interaction. We propose a model for ATP regulation of KaiB-KaiC interaction.

Materials and Methods

Preparation of Kai protein

We produced recombinant Kai proteins derived from the thermophylic cyanobacterium Thermocynechococcus elongatus BP-1 using Escherichia coli as a host, as described previously, with modifications [13]. The plasmids expressing KaiA whose subunit consists of 283 amino acid residues, wild-type KaiB (KaiB_WT; pTekaiB_WT), whose subunit consists of 108 amino acid residues, a mutant KaiB with a C-terminal deletion of residues 95 to 108 (KaiB_1-94; pTekaiB_1-94), wild-type KaiC (KaiC_WT; pTekaiC_WT), whose subunit consists of 518 amino acid residues, and mutant KaiCs— a mutant KaiC with aspartate substitutions at the two phosphorylation sites that is considered to mimic the fully phosphorylated state of KaiC, KaiC_S431D&T432D (KaiC_DD; pTekaiC_DD), a mutant KaiC with alanine substitutions at the two phosphorylation sites that is considered to mimic fully unphosphorylated state of KaiC, KaiC_S431A&T432A (KaiC_AA; pTekaiC_AA), a mutant KaiC with glutamine substitutions at the two deduced catalytic glutamate residues of the C-terminal ATPase motifs that lacks the C-terminal ATPase activity of KaiC, KaiC_CatE2- (pTekaiC_CatE2-), KaiC_CatE2-_/S431A&S432A (KaiC_CatE2-_/AA; pTekaiC_CatE2-_/AA), an N-terminal domain mutant protein of residues 1 to 268 (KaiC_N; pTekaiC_N), and a C-terminal domain mutant protein of residues 269 to 518 with a mutation with aspartate substitutions at the two KaiC phosphorylation sites (KaiC_C/DD; pTekaiC_C/DD)—have been described previously [8,28]. We constructed plasmids for the production of KaiC_{K53H/ DD} (pTekaiC_K53H/DD), which is deficient in the N-terminal ATP binding site (N-terminal ATPase motifs) and carries aspartate substitutions at the two phosphorylation sites on the C-terminal domain (DD mutation), and KaiC_CatE1-_/DD (pTekaiC_CatE1-_/DD) , which is deficient in the N-terminal ATPase and carries the DD mutation, by replacing a 1.25-kb BamHI-EcoRI fragment carrying the 3'-region of each kaiC gene in pTekaiC_K53H and pTekaiC_CatE1-, respectively, with that of pTekaiC_DD. We constructed a plasmid for KaiC_K294H/DD (pTekaiC_K294H/DD), which is deficient in the N-terminal ATP binding site and carries the DD mutation as descried previously [13]. We purified KaiA, KaiB, and KaiC as described previously [28] and stored them at -85 °C until used. KaiA is a dimer [9,19,20], KaiB_WT tetramer [23-25], and KaiB_1-94 dimer [17]. Thus unless otherwise stated, we expressed the concentrations of KaiA, KaiB_WT, KaiB_1-94, KaiC^1mer, and KaiC^6mer as those of a dimer, tetramer, dimer, monomer, and hexamer, respectively.

Preparation of KaiC^6mer

To allow the hexamerization of KaiC, we incubated KaiC^1mer with 1 mM ATP or 1 mM ATP analogs, 5’-adenylylimidodiphosphate (AMPPNP) and adenosine 5'-O-(3-thio) triphosphate (ATPγS), in the presence of 5 mM MgCl₂ in 20 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl at 20 °C for 20 min. We then subjected the reaction mixtures to gel filtration chromatography on a Superdex 200/HR 10/30 column (GE Healthcare) equilibrated with 20 mM HEPES-NaOH buffer (pH 7.5) containing 150 mM NaCl (reaction buffer), 0.1 mM ATP (or ATP analogs) and 5 mM MgCl₂ at 4 °C, and we collected fractions containing KaiC^6mer. We similarly prepared KaiC^6mer in the absence of MgCl₂. Unless otherwise stated, KaiC^6mer means KaiC hexamerized with 1 mM ATP plus 5 mM MgCl₂ (Mg-ATP). The phosphorylation level of the KaiC_WT preparations used here was about 30 %.

Assay for the complex formation of monomeric KaiC (KaiC^1mer) with KaiBs by native polyacrylamide gel electrophoresis (native PAGE)

We incubated reaction mixtures containing 15 μM KaiB_1-94 or 7.5 μM KaiB_WT and 5 μM KaiC^1mer or 2.5 μM KaiC^6mer at 4 °C in reaction buffer and subjected 20-μl aliquots to native PAGE on 10 % gels (acrylamide: bisacrylamide = 37.5: 1) and stained the gels with Coomassie Brilliant Blue (CBB). We estimated the amount of KaiB-KaiC^1mer complex by densitometry using a Lane Analyzer (ATTO, Tokyo, Japan) and a CS Analyzer (ATTO).

Assay for formation of the KaiB_1-94-KaiC^6mer complex by native-PAGE and 2-dimensional sodium dodecyl sulfate (SDS)-PAGE

We incubated reaction mixtures containing 10 μM KaiB_1-94 and 6 μM KaiC^6mer at 4, 25, or 40 °C in reaction buffer and subjected aliquots to native PAGE. We confirmed the presence of KaiB_1-94 and KaiC in the complex bands by native PAGE followed by SDS-PAGE on 18 % gels, as described previously [8].

Assay for formation of the KaiB_1-94-KaiC^1mer complex and KaiB_1-94-KaiC^6mer complex by gel filtration chromatography

To assay for formation of the KaiB_1-94-KaiC^1mer complex and the KaiB_1-94-KaiC^6mer complex, we incubated reaction mixtures containing 36 μM KaiB_1-94 and 12 μM KaiC^1mer in reaction buffer and those containing 7.5 μM KaiB_1-94 and 2.5 μM KaiC^6mer (KaiC_WT or mutant KaiCs) in the buffer with (for KaiC^6mer) or without (for KaiC^1mer) Mg-ATP at 4 °C for 6 h. We then analyzed the mixtures by gel filtration chromatography on a Superdex 200/HR 10/30 column equilibrated with reaction buffer with (for KaiC^6mer) or without (for KaiC^1mer) 0.1 mM ATP and 5 mM MgCl₂ at 4 °C. We subjected all peak fractions to SDS-PAGE and visualized by staining with CBB.

Immunoblot analysis

Because we could not easily detect the KaiB_1-94 contained in a putative complex between KaiB_1-94-KaiC_N^6mer by CBB staining of SDS-PAGE gels, we subjected the gels to immunoblotting. We incubated reaction mixtures containing KaiB_1-94 and/or KaiC_N^6mer at 4 °C for 6 h as described above, and then subjected them to gel filtration chromatography on a Superdex 200/HR 10/30 column equilibrated with reaction buffer containing 0.5 mM ATP and 5 mM MgCl₂ at 4 °C. We subjected fractions containing a putative KaiB_1-94-KaiC_N^6mer complex to SDS-PAGE, blotted the proteins onto Immobilon-P Transfer Membrane (Millipore), and visualized them using the ECL Western Blotting Analysis System (GE Healthcare) with a rabbit anti-KaiB antiserum (diluted to 1/2000) as a primary antibody and a donkey anti-rabbit Ig antibody (GE Healthcare) as a secondary antibody, as described previously [8].

Assay for the time course of formation of the KaiB_1-94-KaiC_DD^1mer complex

We incubated KaiC_DD^1mer (5 μM) with 5 μM KaiB_1-94 in reaction buffer at 4 °C for various periods and subjected aliquots of the reaction mixtures to native PAGE on 10 % gels, staining the gels with CBB as described above. We estimated the amount of KaiB_1-94-KaiC_DD^1mer complex from the intensity of the band by densitometry as described above.

KaiB_1-94-concentration dependence of KaiB-KaiC^1mer complex formation

We incubated 1 μM KaiC_DD^1mer with various amounts of KaiB_1-94 (0.5, 0.75, 1.0, 1.25, 1.5, 2.0, 2.5, 3.0, and 4.0 μM) in reaction buffer at 4 °C for 6 h. Other conditions were the same as described above.

Assay for Mg-ATP-induced dissociation of the KaiB_1-94-KaiC^1mer complex

We assayed the ATP-induced hexamerization of KaiC_DD^1mer in the KaiB_1-94-KaiC^1mer complex and the concomitant release of KaiB_1-94. To obtain the KaiB_1-94-KaiC_DD^1mer complex, we incubated reaction mixtures containing 24 μM KaiB_1-94 and 24 μM KaiC_DD^1mer in reaction buffer at 4 °C for 16 h and then subjected the mixtures to gel filtration chromatography on a Superdex 75/HR 10/30 column (GE Healthcare) equilibrated with reaction buffer at 4 °C. We added 1 mM ATP with or without 5 mM MgCl₂ to the obtained KaiB_1-94-KaiC_DD^1mer complex and then incubated the reaction mixtures in reaction buffer at 4 °C for 6 h. Then, we subjected the reaction mixtures to gel filtration chromatography on a Superdex 200/HR 10/30 column equilibrated with reaction buffer at 4 °C. We analyzed all peak fractions by SDS-PAGE on 18 % gels, and then stained the gels with CBB.

Preparation of KaiC^6mer formed with ADP and aluminum fluoride (KaiC^6mer (ADP-AlF_X)) and assay for the KaiB_1-94-KaiC^6mer (ADP-AlF_X) complex

We incubated KaiC^1mer (20 μM) in reaction buffer containing 6 mM ADP, 30 mM MgCl₂, 2.5 mM NaF, and 2.5 mM AlCl₃ at 25 °C for 2 h to form KaiC^6mer (ADP-AlF_X) [31]. We then mixed the reacion mixtures with an equal volume of 0 or 60 μM KaiB_1-94 in the same buffer and incubated them further at 25 °C for 6 h. We subjected the reaction mixtures to gel filtration chromatography on a Superdex 200/HR 10/30 column equilibrated with reaction buffer at 4 °C. We subjected peak fractions to SDS-PAGE and visualized the proteins by staining with CBB.

Assay for the KaiA-enhanced autophosphorylation of KaiC^6mer

We incubated reaction mixtures containing 0.5 μM KaiC^6mer, 0.5 μM KaiA, and Mg-ATP in reaction buffer at 40 °C for various periods. We then subjected aliquots of the reaction mixtures to SDS-PAGE on 12.5 % gels (acrylamide: bisacrylamide = 144: 1), and stained the gels with CBB. We estimated the amount of the protein from the intensities of bands by densitometry as described above.

Assay for ATPase activity

We measured the ATPase activity of KaiC^6mer using BIOMOL GREEN (Enzo Life Science International, Inc., Farmingdale, New York, USA) according to the supplier's instruction manual. We incubated KaiC^6mer (1 μM) in reaction buffer containing Mg-ATP at 4 °C for 6 h, and then incubated 50-μl aliquots of the reaction mixtures with 50 μl BIOMOL GREEN at 25 °C for 20 min. We measured the absorbance of the samples at 620 nm (A₆₂₀) using an ARVO X4 plate reader (PerkinElmer Inc., Waltham, MA, USA), calculated the amount of Pi released from ATP from triplicate experiments, and expressed the ATPase activity as mol Pi released per mol KaiC^6mer per h at 4 °C. We showed the values after subtracting the buffer background (0.024 ± 0.03 ATP molecules/h).

Results

Association of KaiC^1mer with KaiB_1-94

At 40 °C, KaiB_WT formed a complex with KaiC_DD^6mer (Figure 1B) but the complex formation took more than 9 h to reach a plateau (Figure 1B). KaiB_1-94-KaiC_DD^6mer complex formation, on the other hand, reached a plateau within 6 h with a time (t_1/2) of 1.2 h where the half maximal KaiB_1-94-KaiC^6mer complex formation occurred, and even at 4 °C showed no lag (Figure 1B). Thus, to analyze the formation of the KaiB-KaiC complex in detail, we used KaiB_1-94 and KaiC_DD at 4 °C.

First, we examined KaiC_DD^1mer complex formation with KaiB_1-94 and KaiB_WT. When we incubated 5 μM KaiC_DD^1mer with 15 μM KaiB_1-94 or 7.5 μM KaiB_WT at 4 °C for 6 h, KaiC_DD^1mer formed a complex with KaiB_1-94 (Figure 1C and Table S1) as KaiC_DD^6mer (Figure 1B), whereas it showed only weak complex formation with KaiB_WT (Figure 1D). The complex formation occurred without delay and reached a plateau at 6 h (Figure 1E). The time (t_1/2) where the half maximal KaiB_1-94-KaiC^1mer complex formation occurred at 4 °C was about 2.5 h (Figure 1E). The concentration of KaiB_1-94 where the half maximal KaiB_1-94-KaiC^1mer complex formation occurred was about 0.7 μM (Figure 1F). This value falls within the concentration range at which clock oscillations occur in the in vitro KaiABC clock system [5].

KaiB-interacting domain of KaiC

The KaiC subunit is composed of an N-terminal domain (KaiC_N) and a C-terminal domain (KaiC_C) [32]. When we incubated 5 μM of KaiC_DD monomeric domain protein (KaiC_N^1mer or KaiC_C/DD^1mer) with 30 μM KaiB_1-94 at 4 °C for 6 h, only KaiC_N^1mer formed a complex with KaiB_1-94 (Figures 2A and B, and Table S1). Even when the reaction mixtures were incubated at 25 °C, we did not detect any KaiB_1-94-KaiC_C/DD^1mer complex (Figure 2B). Thus, the KaiB-interacting site of KaiC was located on the N-terminal domain of KaiC.

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Figure 2. Native PAGE gels, gel filtration chromatography elution profiles, and the immunoblots of the KaiB_1-94-KaiC complexes.

A, B. Native PAGE gels. Reaction mixtures containing 15 μM KaiB_1-94 and 5 μM KaiC_N^1mer (A) or KaiC_C/DD^1mer (B) were incubated at 4 °C for the periods indicated (hereafter, unless otherwise stated, 0 h shows data for samples taken at the onset of incubation) and subjected to native PAGE. Other conditions were the same as for Figure 1C legend. C. Gel filtration chromatography elution profiles and an immunoblot. Reaction mixtures containing 7.5 μM KaiB_1-94, 2.5 μM KaiC_N^6mer, and 1 mM ATP plus 5 mM MgCl₂ (Mg-ATP) were incubated at 4 °C for 6 h and then subjected to gel filtration chromatography on a Superdex 200/HR 10/30 column equilibrated with reaction buffer containing 0.5 mM ATP and 5 mM MgCl₂ at 4 °C. We also separately analyzed KaiB_1-94 and KaiC_N^6mer by gel filtration chromatography as controls. The peak fraction samples were subjected to SDS-PAGE, blotted to PVDF membranes, and reacted with an anti-KaiB antiserum. Other conditions were the same as for Figure 1C legend. Black solid line, KaiB_1-94 + KaiC_N^6mer; gray solid line, KaiC_N^6mer; black broken line, KaiB_1-94.

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When we incubated 2.5 μM KaiC_N^6mer with 7.5 μM KaiB_1-94 in the presence of Mg-ATP, KaiC_N^6mer showed a weak association with KaiB_1-94 that was detected by gel filtration chromatography but not by Native PAGE (Figure 2C and Table S2). Unexpectedly, most KaiC_N^6mer became monomeric when it formed a complex with KaiB_1-94 (Figure 2C). KaiC_C/DD^6mer, on the other hand, did not form a complex with KaiB_1-94 (Table S2). KaiC^6mer, therefore, also associates with KaiB_1-94 via its N-terminal domain.

Mg-ATP-induced dissociation of the KaiB_1-94-KaiC^1mer complex

Addition of ATP as well as Mg-ATP was able to hexamerize KaiC_DD^1mer and KaiC_N^1mer (Figure 3A) as described previously [33]. When the KaiB_1-94-KaiC_DD^1mer complex was incubated in the presence of 1 mM ATP or 1 mM ATP plus 5 mM MgCl₂ (Mg-ATP) at 4 °C for 6 h, Mg-ATP, but not ATP, induced the hexamerization of KaiC_DD (Figure 3B) and the concomitant release of KaiB_1-94 (Figures 3B and 3C). We also detected a small amount of the KaiB_1-94-KaiC_DD^6mer complex (Figure 3C), which was likely formed from the KaiC_DD^6mer hexamerized by Mg-ATP, and the KaiB_1-94 released from the KaiB_1-94-KaiC_DD^1mer complex during incubation. These results suggest that Mg-ATP but not ATP reduced the affinity of KaiC^1mer for KaiB_1-94 to dissociate the KaiB_1-94-KaiC_DD^1mer complex. When we examined the ATP- and Mg-ATP-induced oligomerization of KaiC_N in the KaiB_1-94-KaiC_N^1mer complex, we obtained essentially the same results; Mg-ATP, to a much greater extent than ATP, induced oligomerization of KaiC_N in the KaiB_1-94-KaiC_N^1mer complex (Figure 3B). These results suggest that Mg-ATP (and ATP) inhibited the association of KaiB_1-94 with the N-terminal domain of KaiC.

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Figure 3. Mg-ATP-induced dissociation of the KaiB_1-94-KaiC^1mer complex.

A. Native PAGE gels showing the ATP-induced hexamerization of KaiC_DD^1mer and KaiC_N^1mer in the presence or absence of MgCl₂. KaiCs^1mer (5 μM) were hexamerized by incubation with 1 mM ATP with (Mg-ATP) or without (ATP) 5 mM MgCl₂ at 4 °C for the periods indicated. Other conditions were the same as described for Figure 1C legend. B. Native PAGE gels of the KaiB_1-94-KaiC^1mer complex after incubation with ATP or Mg-ATP. Reaction mixtures containing 15 μM KaiB_1-94 and 5 μM KaiC_DD^1mer in reaction buffer were incubated at 4 °C for 16 h to allow the formation of the KaiB_1-94-KaiC^1mer complex. After addition of 1 mM ATP with (Mg-ATP) or without (ATP) 5 mM MgCl₂ to the complex, the reaction mixtures were further incubated in reaction buffer at 4 °C for 6 h. Other conditions were the same as described for Figure 1C. C. Gel filtration chromatography elution profiles of the KaiB_1-94-KaiC_DD^1mer complex incubated with or without Mg-ATP. Reaction mixtures containing 24 μM KaiB_1-94 and 24 μM KaiC_DD^1mer in reaction buffer were incubated at 4 °C for 16 h and then subjected to gel filtration chromatography on a Superdex 75/HR 10/30 column equilibrated with reaction buffer at 4 °C, and KaiB_1-94-KaiC_DD^1mer complex fractions were collected. With (gray) or without (black) addition of Mg-ATP to the complex, the reaction mixtures were further incubated in reaction buffer at 4 °C for 6 h and then subjected to gel filtration chromatography on a Superdex 200/HR 10/30 column equilibrated with reaction buffer. The peak fractions were subjected to SDS-PAGE. Other conditions were the same as described for Figure 1C. Left and right gels, the 1st and 3rd peak fractions of the reaction products with addition of Mg-ATP, respectively; middle gels, the peak fraction products without addition of Mg-ATP corresponding to the 2nd peak fraction of the reaction products with addition of Mg-ATP.

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Effects of mutations in the ATPase motifs of KaiC on KaiB_1-94-KaiC complex formation

Both the N- and C-terminal domains of the KaiC subunit have a series of ATPase motifs (a Walker's motif A, a Walker's motif B, and a CatE [2]). When we examined the effects of mutations in those motifs on formation of KaiB_1-94-KaiC^1mer complexes—using KaiCs with K53H and K294H mutations in Walker’s motif A and CatE1^- and CatE2^- mutations in CatEs [32]—we found that all the mutants we examined (KaiC_K53H/DD, KaiC_CatE1-_/DD, KaiC_K294H/DD, and KaiC_CatE2-_/DD) formed complexes with KaiB_1-94 (Figure 4A and Table S1). Thus, none of the mutations in the ATPase motifs affected formation of the KaiB_1-94-KaiC_DD^1mer complex that occurs in the absence of ATP.

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Figure 4. Effects of mutations in the ATPase motifs and phosphorylation sites of KaiC on formation of the KaiB_1-94-KaiC^6mer complex.

A. Native PAGE gels of the reaction products of KaiC^1mer with KaiB_1-94. Reaction mixtures containing 15 μM KaiB_1-94 and 5 μM KaiC^1mer were incubated at 4 °C for the periods indicated. Other conditions were the same as described for Figure 1. B. Native PAGE gels of the reaction products of KaiC^6mer with KaiB_1-94. Reaction mixtures containing 5 μM KaiB_1-94 and 1 μM KaiC^6mer were incubated in the presence of Mg-ATP at 4 °C for the periods indicated. Other conditions were the same as described for Figure 1C. C. A typical 2D SDS-PAGE gel from the native-PAGE gel shown in Figure 4B. The protein bands were excised, and the proteins were extracted from them and subjected to SDS-PAGE. Other conditions were the same as described for Figure 1C. The bands 1 to 3 were the KaiC_CatE2-_/DD^6mer band (control), the upper band of the reaction products of KaiC_CatE2-_/DD^6mer with KaiB_1-94 incubated at 4 °C for 6 h, and the lower band of the reaction products of KaiC_CatE2-_/DD^6mer with KaiB_1-94 incubated similarly. D. Native PAGE gels of the reaction products of KaiC^6mer with KaiB_1-94. Reaction mixtures were incubated at 4 °C for the periods indicated. Other conditions were the same as described for Figure 4B expect that unphosphorylatable mutant KaiCs were used. E. SDS-PAGE gels showing no phosphorylation of KaiC_AA^6mer, KaiC_CatE2-^6mer, and KaiC_K294H^6mer. KaiCs^6mer (0.5 μM) were incubated with Mg-ATP in the presence of 0.5 μM KaiA at 40 °C for the periods indicated and then subjected to SDS-PAGE. p-KaiC, the phosphorylated forms of KaiC; np-KaiC, the unphosphorylated form of KaiC. Other conditions were the same as described for Figure 3.

https://doi.org/10.1371/journal.pone.0080200.g004

When we examined KaiB_1-94-KaiC^6mer complex formation in the presence of Mg-ATP, both KaiC_K294H/DD^6mer and KaiC_CatE2-_/DD^6mer, which have a C-terminal ATPase motif mutation, as well as KaiC_DD control, formed complexes with KaiB_1-94 whereas KaiC_K53H/DD^6mer and KaiC_CatE1-_/DD^6mer, which have an N-terminal ATPase motif mutation, did not (Figure 4B). Native PAGE followed by SDS-PAGE revealed that all the candidate complex bands examined contained both KaiB_1-94 and KaiC (a typical example is shown in Figure 4C). These results indicate that KaiC’s N-terminal ATPase motifs were responsible for formation of the KaiB_1-94-KaiC^6mer complex that occurred in the presence of Mg-ATP. This observation is consistent with the finding described above that KaiC associates with KaiB_1-94 via its N-terminal domain (Figure 2) and that Mg-ATP (and ATP) inhibits the association (Table S2).

Effects of the ATP, Mg-ATP, Mg-AMPPNP, and Mg-ATPγS used for the hexamerization of KaiC on KaiB_1-94-KaiC^6mer complex formation

The observations described above suggest that ATP hydrolysis followed by the release of ADP from the N-terminal ATPase motifs are involved in KaiC^6mer-KaiB_1-94 complex formation. We therefore examined whether the KaiCs^6mer hexamerized by unhydrolyzable substrate analogs of ATPases, ATP (KaiC^6mer (ATP)), Mg-AMPPNP (KaiC^6mer (Mg-AMPPNP)), and Mg-ATPγS (KaiC^6mer (Mg-ATPγS)) formed a complex with KaiB_1-94 and found that only KaiC^6mer hexamerized with Mg-ATP (KaiC^6mer (Mg-ATP)), which is a hydrolyzable substrate for ATPases, did (Figure 5A). These results support our hypothesis that hydrolysis of ATP in the N-terminal ATPase motifs is required for KaiB_1-94-KaiC^6mer complex formation because ATP and the unhydrolyzable substrates analogs examined inhibited KaiB_1-94-KaiC^6mer complex formation.

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Figure 5. Effects of the ATP and ATP analogs used for the hexamerization of KaiC on formation of the KaiB_1-94-KaiC_DD^6mer complex assayed by native PAGE and gel filtration chromatography.

A. Native PAGE gels. Reaction mixtures were incubated at 4 °C for the periods indicated. Other conditions were the same as described for Figure 4B except that KaiC_DD^6mer (ATP), KaiC_DD^6mer (Mg-AMPPNP), and KaiC_DD^6mer (Mg-ATPγS) were used in the presence of 1 mM ATP, Mg-AMPPNP, and Mg-ATPγS, respectively. B. Gel filtration chromatography elution profiles of KaiC_DD^6mer (Mg-ADP-AlF_X) (dotted line) and a KaiB_1-94-KaiC_DD^6mer (Mg-ADP-AlF_X) complex (solid line). KaiC_DD^1mer (20 μM) was incubated in reaction buffer containing 6 mM ADP, 30 mM MgCl₂, 2.5 mM NaF, and 2.5 mM AlCl₃ at 25 °C for 2 h, and the reaction mixtures were mixed with an equal volume of 0 or 60 μM KaiB_1-94 in the same buffer and then further incubated at 25 °C for 6 h. The reaction mixtures were then subjected to gel filtration chromatography on a Superdex 200/HR 10/30 column equilibrated with reaction buffer at 4 °C. Other conditions were the same as described for Figure 2.

https://doi.org/10.1371/journal.pone.0080200.g005

KaiC^6mer (Mg-ADP-AlF_X) forms a complex with KaiB_1-94

Because ADP-AlF_X mimics ADP-P_i [34], we used gel filtration chromatography to determine whether Mg-ADP-AlF_X hexamerizes KaiC_DD^1mer and confirmed that it did (Figure 5B). Next, we examined the possible complexes formed by KaiC_DD^6mer (Mg-ADP-AlFx) with KaiB_1-94. KaiC_DD^6mer (Mg-ADP-AlF_X) formed a complex with KaiB_1-94 (Figure 5B), but we also detected a KaiB_1-94-KaiC_DD^1mer complex under conditions wherein a substantial amount of KaiC_DD^1mer was not hexamerized (Figure 5B). This observation supports our above conclusion that ATP hydrolysis is required for formation of the KaiB_1-94-KaiC_DD^6mer complex. Therefore, the conformation of KaiC_DD^6mer (Mg-ADP-P_i) that allows complex formation with KaiB_1-94 may differ from that of KaiC_DD^6mer (Mg-ATP) without ATP hydrolysis and that of KaiC^6mer (ATP).

Effects of KaiC phosphorylation-site mutations and autophosphorylation mutations on KaiB_1-94-KaiC^6mer complex formation

We examined the effects of mutations in the two phosphorylation sites of KaiC on KaiB_1-94-KaiC^6mer complex formation. While KaiC_WT^6mer (Figure 4D) and KiaC_DD^6mer (Figure 5A) formed a complex with KaiB_1-94, KaiC_AA^6mer did not form such a complex (Figure 4D). These results are consistent with previous reports showing that phosphorylated KaiC (KaiC_DD and KaiC_DE, a mutant KaiC similar to KaiC_DD) but not unphosphorylated KaiC (KaiC_AA) formed a complex with KaiB_WT [5,16-18]. When KaiC_WT^6mer was incubated with Mg-ATP in the presence of KaiA, it was highly phosphorylated (Figure 4E) [8,9,13,21,27,35]. The two upper bands correspond to the phosphorylated forms of KaiC whereas the lowest band corresponds to the unphosphorylated form of KaiC (Figure 4E) [3,13]. KaiC_AA^6mer, KaiC_CatE2-^6mer, and KaiC_K294H^6mer did not show any phosphorylated bands even in the presence of KaiA (Figure 4E).

Next, we examined the effects of KaiC autophosphorylation mutations on KaiB_1-94-KaiC^6mer complex formation. In spite of lacking the autophosphorylation [13], both KaiC_K294H^6mer and KaiC_CatE2-^6mer formed complexes with KaiB_1-94 (Figure 4D). Thus, phosphorylation of KaiC per se was not essential for KaiB_1-94-KaiC^6mer complex formation, although KaiC’s phosphorylation state might indirectly modulate complex formation.

Effects of KaiC phosphorylation-site mutations on the N-terminal ATPase activity of KaiC

To examine the effects of mutations in the two phosphorylation sites of KaiC on the N-terminal ATPase activity of KaiC, we compared the ATPase activities of KaiC_CatE2-_/AA^6mer, KaiC_CatE2-_/DD^6mer, and KaiC_CatE2-^6mer because KaiC_CatE2- lacks the C-terminal ATPase activity of KaiC [13]. The ATPase activities of KaiC_CatE2-_/DD^6mer and KaiC_CatE2-^6mer reflect the N-terminal ATPase activity while that of KaiC_DD^6mer and KaiC_WT^6mer reflect the total ATPase activity. The former activities were approximately half of the latter activities (Figure 6 and Table S4). These four KaiCs^6mer formed a complex with KaiB_1-94 (Tables S2 and S3). On the other hand, the ATPase activity of KaiC_AA^6mer, which did not form a complex with KaiB_1-94 (Table S3), showed 6 times higher ATPase activity than KaiC_DD^6mer and KaiC_WT^6mer (Figure 6). In consistent with this, KaiC_AA has been reported to show 2.5 times higher ATPase activity than KaiC_DE, which is a mutant KaiC similar to KaiC_DD [4]. The ATPase activity of KaiC_CatE2-_/AA, which formed a complex with KaiB_1-94 that could only be detected by silver staining (Table S3), was more than 3 times as high as those of KaiC_CatE2-_/DD^6mer and KaiC_CatE2-^6mer. The ATPase activities of KaiC_CatE2-_/AA^6mer, KaiC_CatE2-_/DD^6mer, and KaiC_CatE2-^6mer all reflect the N-terminal ATPase activity of KaiC. Thus, although the N-terminal ATPase activity of KaiC is probably required for KaiC^6mer to form a complex with KaiB_1-94, its excessively high activity (KaiC_AA^6mer and KaiC_CatE2-_/AA^6mer) may inhibit complex formation. The ATPase activity of KaiC_CatE1-_/DD^6mer reflecting the C-terminal ATPase activity of KaiC was almost the same as that of KaiC_C/DD^6mer and approximately half of that of KaiC_DD^6mer (Figure 6 and Table S4), as described previously [10].

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Figure 6. Effects of mutations in the ATPase motifs and phosphorylation sites of KaiC on KaiC’s ATPase activity.

We incubated 1 μM KaiCs^6mer with Mg-ATP in reaction buffer at 4 °C for 6 h and then measured ATPase activities. Values are means ± SD from triplicate assay. KaiCs^6mer: WT, KaiC_WT^6mer; DD, KaiC_DD^6mer; AA, KaiC_AA^6mer; N, KaiC_N^6mer; C/DD, KaiC_C/DD^6mer; K53H/DD, KaiC_K53H/DD^6mer; CatE1^-/DD, KaiC_CatE1-_/DD^6mer; CatE2^-, KaiC_CatE2-^6mer; CatE2^-/AA, KaiC_CatE2-_/AA^6mer; CatE2^-/DD, KaiC_CatE2-_/DD^6mer; K294H/DD, KaiC_K294H/DD^6mer.

https://doi.org/10.1371/journal.pone.0080200.g006

Discussion

Since KaiC^1mer formed a complex with KaiB_1-94 (Figure 1C), the KaiC subunit per se is able to form a complex with KaiB. Recently, NMR analysis revealed that KaiC^1mer forms a complex with KaiB via its N-terminal domain [29], and here KaiC (KaiC^6mer as well as KaiC^1mer) formed a complex with KaiB_1-94 via its N-terminal domain (Figure 2). ATP, Mg-ATP, Mg-AMPPNP, and Mg-ATPγS hexamerize KaiC, probably by binding subunits [13,32,36,37]. KaiC^6mer (ATP), KaiC^6mer (Mg-AMPPNP), and KaiC^6mer (Mg-ATPγS) did not form a complex with KaiB_1-94 (Figure 5A). This indicates that when KaiC-bound ATP (or ATP analogs) is unhydrolyzable, KaiC does not form a complex with KaiB. KaiC_DD^6mer (Mg-ADP-AlF_X), which mimics the Mg-ADP-Pi state of KaiC_DD^6mer, formed a complex with KaiB_1-94 (Figure 5B). It is likely, therefore, that ATP regulates KaiB-KaiC^6mer complex formation by hindering complex formation by KaiC^6mer-bound ATP, and its hydrolysis is required for complex formation.

That mutations in the N-terminal but not its C-terminal ATPase motifs affected the complex formation of KaiC^6mer (Mg-ATP) with KaiB_1-94 (Figure 4B) indicates that ATP hydrolysis by the N-terminal KaiC’s ATPase motifs is responsible for KaiB-KaiC^6mer complex formation. These results are consistent with a recent report [38]. Because the KaiC_N^6mer-KaiB_1-94 complex rapidly dissociated into a KaiB_1-94-KaiC_N^1mer complex (Figure 2C), the partial dissociation (or relaxation) of the N-terminal domains of KaiC^6mer probably occurred on the interaction of KaiC^6mer with KaiB. This partial dissociation of the KaiC^6mer N-terminal domains is likely required for formation of KaiB-KaiC^6mer complex via KaiC’s N-terminal domain. The relaxation of the KaiC^6mer N-terminal domains on interaction of KaiB has been revealed recently by NMR analysis [29].

The KaiB molecule, which is a homotetramer organized as a dimer of dimers (KaiB^4mer) [24], probably dissociates into two dimers (KaiB^2mer) on interaction with KaiC^6mer and forms a complex comprising one molecule of KaiC^6mer and two molecules of KaiB^2mer [17], as suggested by cryo-electron microscopy analysis [25]. We have proposed that the positively charged cleft (PC) of the KaiB^4mer molecule, where the functionally important KaiB residues are concentrated, is an active site(s) required for interaction with KaiA and KaiC [24,28,39]. The PC of KaiB^4mer, which is located on the dimer-dimer interface [24], is probably exposed by dissociation of KaiB^4mer into dimers to interact with KaiC^6mer [28,39], whereas the corresponding region of KaiB_1-94, a dimeric mutant of KaiB, is always exposed [17]. Two areas on KaiC^6mer molecule, on the other hand, are highly negatively charged—one around and inside the pore of KaiC^6mer N-terminal domains and the other around the inter-subunit interface of one of two adjacent KaiC^6mer N-terminal domains (Figures S1A and S1B) [37]. Interestingly, the ATP bound to the N-terminal ATPase motifs (namely, ATP-binding sites) is located adjacent to the latter area of KaiC (Figure S1B) [37]. Electrostatic interaction between the PC on KaiB and the aforementioned area of KaiC may allow sequestration of KaiB^4mer (also KaiB^2mer such as KaiB_1-94) and induce dissociation into dimers (temporal weak association). Then, the dissociation of two adjacent N-terminal domains in KaiC^6mer resulting from the hydrolysis of ATP bound to the N-terminal ATPase motifs on one of the two adjacent subunits (Figures S1C and S1D), which pastes the two N-terminal domains each other [13,32,36,37], may expose the latter area of KaiC—a possible KaiB-interacting surface—to KaiB^2mer, and electrostatic interaction between the PC on KaiB and the latter area of KaiC may result in the tight association of KaiB^2mer with KaiC^6mer. The ATP bound to the N-terminal ATPase motifs inhibits the association of KaiB^2mer with KaiC^6mer via KaiC N-terminal domains, as demonstrated in KaiB_1-94-KaiC^6mer complex formation (Figure 3C). Thus, we calculated the surface potentials of KaiC^6mer without ATP (Figure S1C) and with ATP (Figure S1D) and found them to be almost the same and unlikely to affect the interaction of KaiC with KaiB. While KaiB_WT^4mer formed a complex formation with KaiC^6mer (Figure 1B), it did so only slightly with KaiC^1mer (Figure 1D). KaiB_1-94, in contrast, formed a complex with both KaiC^6mer and KaiC^1mer (Figures 1B, 1C and 5A). Therefore, interaction with KaiC^6mer but not with KaiC^1mer likely enhanced KaiB^4mer dimerization, suggesting the possibility that enhancement requires the hexameric structure of KaiC^6mer N-terminal domains.

It has been previously proposed that the phosphorylation state of KaiC was involved in its forming a complex with KaiB [18]. However, our data described here showing that KaiC_K294H^6mer and KaiC_CatE2-^6mer, which lack the autokinase activity (Figure 4E) [13], formed complexes with KaiB_1-94 (Figure 4D) indicated that the phosphorylated state of KaiC is not essential for KaiB-KaiC^6mer complex formation. Our results are consistent with the recently reported results that KaiC_CatE2-^6mer formed a complex with KaiB_WT [38]. The phosphorylation state of KaiC, therefore, is not directly involved in and essential for complex formation.

KaiC_AA^6mer and KaiC_CatE2-_/AA^6mer, which did not form a complex with KaiB_1-94, showed much higher ATPase activity than any other KaiC ATPase motif mutants we examined (Figure 6 and Table S4). The excessively high ATPase activity of the N-terminal ATPase motifs of KaiC_AA^6mer, which bounces in and out of the ATP bound to the N-terminal ATPase motifs, may hinder formation of the KaiB-KaiC^6mer complex. In KaiC^6mer, the phosphorylation state of the C-terminal domain could affect its association with KaiB via the N-terminal domain through modulating the N-terminal ATPase activity. However, we cannot exclude a possibility that KaiC_AA^6mer, which is likely not a perfect mimic for the fully unphosphorylated form of KaiC, might have a changed structure, which might enhance its ATPase activity but might reduce its association with KaiB. We propose the following model for ATP regulation of KaiB-KaiC interaction. The KaiC subunit is able to form a complex with KaiB (Figure 7A). The N-terminal domains of KaiC^6mer are partially dissociated (relaxed) when the ATP bound to the N-terminal ATPase motifs that pastes adjacent N-terminal domains each other in KaiC^6mer is hydrolyzed (Figure 7B), which allows KaiB to associate with the KaiC^6mer N-terminal domains. Then, KaiC^6mer-associated KaiB suppresses the ATPase activity of KaiC^6mer [4] by inhibiting ATP binding to KaiC^6mer N-terminal domains. KaiC_AA^6mer and KaiC_CatE2-_/AA^6mer, which seem to mimic the unphosphorylation state of KaiC, have excessively high N-terminal ATPase activity (Figure 6 and Table S4), and that may cause rapid interconversion of the ridged (ATP-bound) and relaxed (ATP-hydrolyzed; ADP-bound or unbound) conformations of the N-terminal domains in KaiC^6mer. We propose here that this rapid interconversion inhibits KaiB-KaiC^6mer complex formation though we cannot explain this inhibiting mechanism at present (Figure 7C).

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Figure 7. ATP-mediated regulation model for KaiB-KaiC interaction.

A. Interaction between KaiB and KaiC^1mer. B. Partial dissociation (relaxation) of the N-terminal domain of KaiC^6mer and complex formation of one KaiC^6mer molecule with 2 KaiB^2mer molecules. For simplification, we express Mg-chelated ATP and ADP as ATP and ADP. The nucleotide state of the C-terminal ATP-binding site (ATPase motifs) of KaiC^6mer is not known. C. Rapid interconversion between the rigid ATP-bound and relaxed ATP-hydrolyzed form (ADP-bound or unbound) conformations in the N-terminal domains of KaiC^6mer.

https://doi.org/10.1371/journal.pone.0080200.g007

The C-terminal ATPase motifs of KaiC are involved in the hexamerization of KaiC C-terminal domains [13] as well as their inter-subunit autophosphorylation [8] and probably autodephosphorylation [12]. Although the KaiC N-terminal ATPase motifs are involved in the hexamerization of KaiC N-terminal domains [13], and the affinity of the N-terminal ATPase motifs for ATP is higher than that of its C-terminal ATPase motifs [13], and therefore, the N-terminal domains are likely to be more tightly connected than the C-terminal domains [13,16,29], the function of the N-terminal ATPase motifs remains unknown. In this investigation, we have succeeded in revealing that the nucleotide state of the N-terminal ATPase motifs regulates KaiB-KaiC interaction. Because KaiB and SasA competitively associate with KaiC via KaiC N-terminal domains [14,17], the nucleotide state also can regulate KaiC-SasA interaction via the KaiB-KaiC interaction. ATP acts not only as a biological fuel, but also as a physiological regulator. There are some examples for ATP regulation of the physiological function. Many different cell types release ATP in response to mechanical or biochemical stimulation, and the released ATP modulates cell function by activating nearby purinoceptors, such as ion channel P2X receptors and G-protein-coupled P2Y receptors [40-42].

Supporting Information

Figure S1.

Electrostatic surface potential of KaiC^6mer and KaiC^1mer of Synechococcus KaiC (PDB code: 2GBL). We calculated electrostatic surface representations of KaiC^6mer (A, B) and KaiC^1mer (C, D) using the PyMOL plug-in APBS [43]. A. Top view of the N-terminal domain of KaiC^6mer with ATP. B. Side view of KaiC^6mer with ATP. Interface of KaiC without ATP (C) and with ATP (D). The saturation thresholds were -5 and +5. For electrostatic surface potential: blue, positive; red, negative. Arrows indicated the negatively charged areas.

https://doi.org/10.1371/journal.pone.0080200.s001

(TIF)

Table S1.

Complex formation of KaiCs^1mer with KaiB_1-94.

https://doi.org/10.1371/journal.pone.0080200.s002

(DOC)

Table S2.

Formation of KaiB_1-94-KaiCs^6mer complex.

https://doi.org/10.1371/journal.pone.0080200.s003

(DOC)

Table S3.

Complex formation of unphosphorylatable KaiCs^6mer with KaiB_1-94.

https://doi.org/10.1371/journal.pone.0080200.s004

(DOC)

Table S4.

Effects of mutations in the ATPase motifs and phosphorylation sites on KaiC ATPase activity.

https://doi.org/10.1371/journal.pone.0080200.s005

(DOC)

Acknowledgments

We thank Satoko Ogawa and Kumiko Tanaka for technical support and Miriam Bloom (SciWrite Biomedical Writing & Editing Services) for professional editing. We also thank Drs. Reiko Murakami and Tomoko Miyata for technical advice.

Author Contributions

Conceived and designed the experiments: RM MI. Performed the experiments: RM AN SY KO. Analyzed the data: RM MI. Contributed reagents/materials/analysis tools: RM MI. Wrote the manuscript: RM.

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Figures

Abstract

Introduction

Materials and Methods

Preparation of Kai protein

Preparation of KaiC6mer

Assay for the complex formation of monomeric KaiC (KaiC1mer) with KaiBs by native polyacrylamide gel electrophoresis (native PAGE)

Assay for formation of the KaiB1-94-KaiC6mer complex by native-PAGE and 2-dimensional sodium dodecyl sulfate (SDS)-PAGE

Assay for formation of the KaiB1-94-KaiC1mer complex and KaiB1-94-KaiC6mer complex by gel filtration chromatography

Immunoblot analysis

Assay for the time course of formation of the KaiB1-94-KaiCDD1mer complex

KaiB1-94-concentration dependence of KaiB-KaiC1mer complex formation

Assay for Mg-ATP-induced dissociation of the KaiB1-94-KaiC1mer complex

Preparation of KaiC6mer formed with ADP and aluminum fluoride (KaiC6mer (ADP-AlFX)) and assay for the KaiB1-94-KaiC6mer (ADP-AlFX) complex

Assay for the KaiA-enhanced autophosphorylation of KaiC6mer

Assay for ATPase activity

Results

Association of KaiC1mer with KaiB1-94

KaiB-interacting domain of KaiC

Mg-ATP-induced dissociation of the KaiB1-94-KaiC1mer complex

Effects of mutations in the ATPase motifs of KaiC on KaiB1-94-KaiC complex formation

Effects of the ATP, Mg-ATP, Mg-AMPPNP, and Mg-ATPγS used for the hexamerization of KaiC on KaiB1-94-KaiC6mer complex formation

KaiC6mer (Mg-ADP-AlFX) forms a complex with KaiB1-94

Effects of KaiC phosphorylation-site mutations and autophosphorylation mutations on KaiB1-94-KaiC6mer complex formation

Effects of KaiC phosphorylation-site mutations on the N-terminal ATPase activity of KaiC

Discussion

Supporting Information

Figure S1.

Table S1.

Table S2.

Table S3.

Table S4.

Acknowledgments

Author Contributions

References

Preparation of KaiC^6mer

Assay for the complex formation of monomeric KaiC (KaiC^1mer) with KaiBs by native polyacrylamide gel electrophoresis (native PAGE)

Assay for formation of the KaiB_1-94-KaiC^6mer complex by native-PAGE and 2-dimensional sodium dodecyl sulfate (SDS)-PAGE

Assay for formation of the KaiB_1-94-KaiC^1mer complex and KaiB_1-94-KaiC^6mer complex by gel filtration chromatography

Assay for the time course of formation of the KaiB_1-94-KaiC_DD^1mer complex

KaiB_1-94-concentration dependence of KaiB-KaiC^1mer complex formation

Assay for Mg-ATP-induced dissociation of the KaiB_1-94-KaiC^1mer complex

Preparation of KaiC^6mer formed with ADP and aluminum fluoride (KaiC^6mer (ADP-AlF_X)) and assay for the KaiB_1-94-KaiC^6mer (ADP-AlF_X) complex

Assay for the KaiA-enhanced autophosphorylation of KaiC^6mer

Association of KaiC^1mer with KaiB_1-94

Mg-ATP-induced dissociation of the KaiB_1-94-KaiC^1mer complex

Effects of mutations in the ATPase motifs of KaiC on KaiB_1-94-KaiC complex formation

Effects of the ATP, Mg-ATP, Mg-AMPPNP, and Mg-ATPγS used for the hexamerization of KaiC on KaiB_1-94-KaiC^6mer complex formation

KaiC^6mer (Mg-ADP-AlF_X) forms a complex with KaiB_1-94

Effects of KaiC phosphorylation-site mutations and autophosphorylation mutations on KaiB_1-94-KaiC^6mer complex formation