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

<div><p>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 <i>in vitro</i>. 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 <i>via</i> 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. </p> </div

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

<p>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. </p

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

<p>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.</p

ATP-mediated regulation model for KaiB-KaiC interaction.

<p>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.</p

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

<p>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. </p

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

<p>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.</p

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

<p>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.</p

Phase resetting of the circadian clock in the <i>b16</i> mutant.

<p>Bioluminescence rhythms of 5-day-old spot cultures of WT and <i>b16</i> cells on HS agar media in white 96-well plates were measured. <b>(A)</b> Bioluminescence trace of a phase shifting experiment. Cells were exposed to a 12 h dark/12 h white light to entrain their circadian clock, and then bioluminescence rhythms were monitored in DD. 5 min light pulses were given at the 34 h time point in DD (arrows). Bioluminescence data are detrended by dividing by the 24 h moving average, and are shown as the mean ± SD of 10 independent cultures. <b>(B)</b> The amount of phase shift. Mean ± SD of the 10 independent cultures of the phase difference (h) between light-pulsed and dark control samples are shown. * <i>P</i> < 0.001 (Student’s <i>t</i>-test). <b>(C)</b> Schematic view of light conditions of the re-entrainment experiment. Cells were entrained by the first LD cycle of white light, and then re-entrained to a 5 h phase advanced LD cycles of red, blue, and violet light. Bioluminescence was monitored after release into DD. <b>(D)</b> Bioluminescence trace of re-entrainment experiment. Data are detrended by dividing by the 24 h moving average, and are shown as the mean ± SD of 10 independent cultures. <b>(E)</b> The amount of phase shift. The mean ± SD of 10 independent cultures of the phase difference (h) between light-entrained and dark control samples is shown. * <i>P</i> < 0.001 (Student’s <i>t</i>-test).</p

Expression analysis of CSL protein.

<p><b>(A)</b> Western blot analysis of CSL-HA in LD and after light pulse. Cell were harvested from LD-entrained HS liquid cultures at midday and midnight. A red light pulse (2 μmol∙m^-2∙s^-1, 0.5 min) was given at midnight, and cells were harvested at times indicated in the graph. 2 μg of total protein was loaded in each lane. <b>(B)</b> Western blot analysis of CSL-HA in a circadian condition. Mid-log-phase (approximately 2 x 10⁶ cells/mL) HS liquid cultures at 24°C were transferred to 12 h darkness at 17°C to synchronize the circadian clock, and then released into a continuous light condition (2 μmol∙m^-2∙s^-1, white light, 17°C). 10 μg of total protein was loaded in each lane. Western blots for α-tubulin are shown as a reference protein (A,B). <b>(C)</b> Immunocytochemical staining of CSL-HA. Cells prepared as described in (A) were used. Cells were counterstained with NPC antibody and 4′,6-diamidino-2-phenylindole (DAPI). Photographs obtained under the same settings of microscope and detector are shown. <i>csl</i> mutant was used as negative controls (A,B,C).</p

Wavelength dependency of the acute light response of ROC15-LUC.

<p>Asynchronous TAP cultures of WT and <i>b16</i> cells in black 24-well plates were subjected to darkness for 3 h for accumulation of ROC15-LUC, and then light pulses were administered. <b>(A)</b> Representative traces of the response to various light qualities. 5 min light pulses were administered by a tunable monochromatic light source. Each monochromatic light was given at 0.5 μmol∙m^-2∙s^-1 at the surface of the culture plates (arrows). Note that the 360 nm light (*) was weaker than those of other wavelengths at the sample level because the lid of culture plates blocked 30% of 360 nm light. The bioluminescence level just before light pulse was set to 100. <b>(B)</b> Sensitivities of ROC15-LUC to light. The sensitivity was represented as the relative bioluminescence level lost after light pulse at the time point when the minimum bioluminescence was detected. Each point represents the mean ± SD of 4–6 independent cultures. * <i>P</i> < 0.05, ** <i>P</i> < 0.01 (Student’s <i>t</i>-test).</p