ATP hydrolysis by KaiC promotes its KaiA binding in the cyanobacterial circadian clock system

Abstract

The cyanobacterial clock is controlled via the interplay among KaiA, KaiB, and KaiC, which generate a periodic oscillation of KaiC phosphorylation in the presence of ATP. KaiC forms a homohexamer harboring 12 ATP-binding sites and exerts ATPase activities associated with its autophosphorylation and dephosphorylation. The KaiC nucleotide state is a determining factor of the KaiB-KaiC interaction; however, its relationship with the KaiA-KaiC interaction has not yet been elucidated. With the attempt to address this, our native mass spectrometric analyses indicated that ATP hydrolysis in the KaiC hexamer promotes its interaction with KaiA. Furthermore, our nuclear magnetic resonance spectral data revealed that ATP hydrolysis is coupled with conformational changes in the flexible C-terminal segments of KaiC, which carry KaiA-binding sites. From these data, we conclude that ATP hydrolysis in KaiC is coupled with the exposure of its C-terminal KaiA-binding sites, resulting in its high affinity for KaiA. These findings provide mechanistic insights into the ATP-mediated circadian periodicity.

Figure 1.. KaiA–KaiC interaction depends on ATP hydrolysis.

(A–D) Native mass spectra of (A, B) KaiC_AA and (C, D) 6:3 mixtures of KaiC_AA and KaiA in the presence of (A, C) 1 mM AMPPNP or (B, D) 1 mM ATP. After 5 h of incubation at 37°C with ATP or AMPPNP, the KaiC solutions with or without KaiA were immediately analyzed by nanoflow electrospray ionization MS. The blue and purple circles show the ion series of the KaiC_AA homohexamer, whereas the orange and red circles show the 2:6 KaiA–KaiC_AA hetero-octamer complexes. See Tables 1 and 2 for assignment details.

Figure S1.. Native MS characterization of KaiA–KaiC_DD interaction with ATP or AMPPNP.

(A–D) Native mass spectra of (A, B) KaiC_DD and (C, D) 6:3 mixtures of KaiC_DD and KaiA in the presence of (A, C) 1 mM AMPPNP or (B, D) 1 mM ATP. After 5 h of incubation at 37°C with the nucleotides, the KaiC solutions with or without KaiA were immediately analyzed by nanoflow electrospray ionization MS. The blue and purple circles show the ion series of the KaiC_DD homohexamer, whereas the red circles show the 2:6 KaiA–KaiC_AA hetero-octamer complexes. See Tables 1 and 2 for assignment details.

Figure S2.. Native MS characterization of KaiC_AA nucleotide state depending on external ATP/ADP condition.

(A–F) Native mass spectra of KaiC_AA mediated by (A–C) ATP and (D–F) AMPPNP. The KaiC_AA hexamers incubated for 5 h at 37°C under (A, D) 100:0, (B, E) 75:25, and (C, F) 50:50 ATP/ADP conditions were immediately analyzed by nanoflow electrospray ionization MS. The blue and purple circles show the ion series of the KaiC_AA hexamers containing seven ATP/five ADP molecules and 12 AMPPNP molecules, respectively.

Figure S3.. Native MS characterization of nucleotide states of the CI and CII domains on KaiC_AA.

(A–C) Native mass spectra of KaiC_AA mediated by ATP after trypsin digestion. After 5 h of incubation at 37°C in the presence of 1 mM ATP, KaiC_AA was buffer-exchanged into 150 mM aqueous ammonium acetate and digested by 0.02 mg/ml trypsin for (A) 0 min, (B), 30 min, and (C) 60 min. The reaction mixture was directly analyzed by nanoflow electrospray ionization MS. The blue circles show the ion series of the KaiC_AA homohexamer containing seven ATP and five ADP molecules, whereas the black circles show the hexameric CI domain (M1–S253) containing six ATP molecules. The CII domain was hardly detected as hexamer under the condition used here.

Figure S4.. Native MS characterization of the KaiA–KaiC_AA/E77Q interaction.

(A–D) Native mass spectra of (A) KaiC_AA/E77Q and (B) a 6:3 mixtures of KaiC_AA/E77Q and KaiA. After 5 h of incubation at 37°C in the presence of 1 mM ATP, the KaiC_AA/E77Q solutions with or without KaiA were immediately analyzed by nanoflow electrospray ionization MS. The blue circles show the ion series of the KaiC_AA/E77Q homohexamer, whereas the red circles show the 2:6 KaiA–KaiC_AA/E77Q hetero-octamer complexes. See Table 2 for assignment details.

Figure S5.. Native MS analysis of KaiC_AA-KaiA complex formation.

(A–E) Native mass spectra of (A) KaiC_AA and (B) 6:1, (C) 6:3, (D) 6:6, and, (E) 6:9 mixtures of KaiC_AA and KaiA in the presence of 1 mM ATP. After 5 h of incubation at 37°C in the presence of 1 mM ATP, the KaiC solutions with or without KaiA were immediately analyzed by nanoflow electrospray ionization MS. The blue circles show the ion series of the KaiC_AA homohexamer, whereas the red and green circles show the 2:6 and 4:6 KaiA–KaiC_AA complexes, respectively.

Figure S6.. ¹H-¹⁵N HSQC spectra of KaiC_AA and its C-terminally truncated mutant.

(A–D) ¹H-¹⁵N HSQC spectra of (A, B) KaiC_AA and (C, D) the mutated KaiC_AA lacking the C-terminal segment 487–518 in the presence of (A, C) 1 mM AMPPNP and (B, D) 1 mM ATP.

Figure 2.. ATP hydrolysis–dependent conformational change of the C-terminal KaiA-binding region of KaiC.

(A–C) ¹H-¹⁵N HSQC spectrum of KaiC_AA in the presence of (A) AMPPNP, (B) ATP, and (C) KaiA and ATP. NMR experiments were set up to take a total time of 3 h using the KaiC hexamer incubated with AMPPNP or ATP for 9 h. Assignments of the peaks from the C-terminal region are given in each spectrum. (D) Plot of relative peak intensity for KaiC_AA resonances in the presence of AMPPNP versus ATP. (E) Plot of relative peak intensity for KaiC_AA resonances in the presence versus absence of KaiA under the ATP condition. In (D) and (E), the residues that yielded no observable peaks under the AMPPNP condition are highlighted in red, whereas the asterisks indicate the proline residues and residues whose chemical shift perturbation data could not be obtained because of severe peak overlapping. (F) Crystal structure of two KaiC protomers in cartoon and surface representation, respectively, in the KaiC homohexameric ring mediated by AMPPNP (PDB ID code: 4O0M). In the crystal structure,the C-terminal region comprises a U-shaped A-loop (Glu487-Ile497) (orange) and a solvent-exposed C-tail (S498-S518), in which only the Ser498-Glu504 part (green) was modeled. The three residues (i.e., Gly488, Ile489, and Ile497) located in the A-loop, whose HSQC peaks were unobserved under the AMPPNP condition, are colored blue. The A-loop and AMPPNP molecule (red) are mediated by a loop comprising residues 415–430 (termed 422-loop, magenta).

Figure 3.. The “fishing a line” mechanism coupling ATP hydrolysis and KaiA-mediated up-regulation of autophosphorylation in the KaiC hexamer.

(A) While both CI and CII domains harbor nucleotide-binding sites and ATPase-active sites at the subunit interfaces, the autokinase activity is exerted only in the CII domain. This is because the autophosphorylation sites (i.e., Ser431 and Thr432) are spatially proximal to the ATP molecule accommodated in the CII domain of the neighboring protomer. (B) In the CII AAA+ ring hexamer, ATP hydrolysis releases the A-loop, which thereby becomes reactive with KaiA. KaiA binding to the C-terminal segments of KaiC facilitates ADP release and ATP incorporation. The rapid ATP/ADP turnover leads to the up-regulation of autophosphorylation of KaiC.