Article Figures & Data
Figures
- Figure 1. KaiA–KaiC interaction depends on ATP hydrolysis.
(A–D) Native mass spectra of (A, B) KaiCAA and (C, D) 6:3 mixtures of KaiCAA 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 KaiCAA homohexamer, whereas the orange and red circles show the 2:6 KaiA–KaiCAA hetero-octamer complexes. See Tables 1 and 2 for assignment details.
- Figure S1. Native MS characterization of KaiA–KaiCDD interaction with ATP or AMPPNP.
(A–D) Native mass spectra of (A, B) KaiCDD and (C, D) 6:3 mixtures of KaiCDD 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 KaiCDD homohexamer, whereas the red circles show the 2:6 KaiA–KaiCAA hetero-octamer complexes. See Tables 1 and 2 for assignment details.
- Figure S2. Native MS characterization of KaiCAA nucleotide state depending on external ATP/ADP condition.
(A–F) Native mass spectra of KaiCAA mediated by (A–C) ATP and (D–F) AMPPNP. The KaiCAA 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 KaiCAA 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 KaiCAA.
(A–C) Native mass spectra of KaiCAA mediated by ATP after trypsin digestion. After 5 h of incubation at 37°C in the presence of 1 mM ATP, KaiCAA 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 KaiCAA 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–KaiCAA/E77Q interaction.
(A–D) Native mass spectra of (A) KaiCAA/E77Q and (B) a 6:3 mixtures of KaiCAA/E77Q and KaiA. After 5 h of incubation at 37°C in the presence of 1 mM ATP, the KaiCAA/E77Q solutions with or without KaiA were immediately analyzed by nanoflow electrospray ionization MS. The blue circles show the ion series of the KaiCAA/E77Q homohexamer, whereas the red circles show the 2:6 KaiA–KaiCAA/E77Q hetero-octamer complexes. See Table 2 for assignment details.
- Figure S5. Native MS analysis of KaiCAA-KaiA complex formation.
(A–E) Native mass spectra of (A) KaiCAA and (B) 6:1, (C) 6:3, (D) 6:6, and, (E) 6:9 mixtures of KaiCAA 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 KaiCAA homohexamer, whereas the red and green circles show the 2:6 and 4:6 KaiA–KaiCAA complexes, respectively.
- Figure S6. 1H-15N HSQC spectra of KaiCAA and its C-terminally truncated mutant.
(A–D) 1H-15N HSQC spectra of (A, B) KaiCAA and (C, D) the mutated KaiCAA 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 KaiCAA 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 KaiCAA resonances in the presence of AMPPNP versus ATP. (E) Plot of relative peak intensity for KaiCAA 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.
Tables
- Table 1.
Summary of native MS characterization of KaiC and KaiA–KaiC complex formed in the presence of AMPPNP.
Figure number Ion series Assignment Theoretical mass (D) Experimental mass (D) Δm (D)a Relative quantity (%) Protein complex AMPPNP number Mg2+ number Fig 1A Blue KaiCAA6 12 12 353,850 353,857 ± 10 −7 — Fig 1C Blue KaiCAA6 12 12 353,850 353,855 ± 9 −5 — Fig 1C Red KaiCAA6/KaiA2 11 12 418,838 418,896 ± 27 −58 53b Fig 1C Orange KaiCAA6/KaiA2 10 12 418,332 418,412 ± 28 −80 47b Fig S2A Blue KaiCDD6 12 12 354,396 354,375 ± 12 21 — Fig S2C Blue KaiCDD6 12 12 354,396 354,447 ± 16 −51 — Fig S2C Red KaiCDD6/KaiA2 12 12 419,890 420,076 ± 47 −186 — - Table 2.
Summary of native MS characterization of KaiC and KaiA–KaiC complex formed in the presence of ATP.
Figure number Ion series Assignment Theoretical mass (D) Experimental mass (D) Δm (D)a Relative quantity (%) Protein complex ATP number ADP number Mg2+ number Fig 1B Blue KaiCAA6 7 5 12 353,462 353,476 ± 18 −14 56b Fig 1B Purple KaiCAA6 7 3 12 352,608 352,593 ± 16 15 44b Fig 1D Blue KaiCAA6 7 5 12 353,462 353,461 ± 14 1 — Fig 1D Red KaiCAA6/KaiA2 6 5 12 418,449 418,445 ± 20 4 67b Fig 1D Orange KaiCAA6/KaiA2 0 11 12 417,969 417,963 ± 20 6 33b KaiCAA6/KaiA2 5 5 12 417,942 417,963 ± 20 −21 Fig S1B Blue KaiCDD6 11 0 12 353,901 353,902 ± 11 −1 — KaiCDD6 6 6 12 353,928 353,902 ± 11 26 Fig S1D Blue KaiCDD6 6 6 12 353,928 353,924 ± 9 4 74b KaiCDD6 11 0 12 353,901 353,924 ± 9 −23 Fig S1D Purple KaiCDD6 6 5 12 353,501 353,477 ± 9 24 26b KaiCDD6 0 12 12 353,448 353,477 ± 9 −29 Fig S1D Red KaiCDD6/KaiA2 2 10 12 419,102 419,102 ± 32 0 — KaiCDD6/KaiA2 7 4 12 419,075 419,102 ± 32 −27 Fig S4A Blue KaiCAA/E77Q6 7 5 12 353,396 353,383 ± 13 13 — Fig S4B Blue KaiCAA/E77Q6 12 0 12 353,796 353,766 ± 29 30 — Red KaiCAA/E77Q6/KaiA2 7 4 12 418,533 418,559 ± 59 −26 —
