From primordial clocks to circadian oscillators – Nature

Construct of KaiC and KaiB expression vectors

The wild-type KaiC_RS (GenBank identifier: ACM04290.1) and KaiB_RS (GenBank: WP_002725098.1) from R. sphaeroides strain KD131/KCTC 12085 (equivalent: Cereibacter sphaeroides strain KD131) constructs used in this paper were ordered from GenScript (Supplementary Table 1). Codon-optimized plasmids for KaiC_RS and KaiB_RS were subcloned into NcoI/KpnI sites of the pETM-41 vector. A QuikChange II Site-Directed Mutagenesis kit (Agilent Technologies) was used to generate single mutant, double mutant and truncated versions of KaiC_RS. The truncated KaiC_RS (KaiC_RS-Δcoil and KaiCI_RS) were generated by introducing stop codons in the KaiC_RS wild-type plasmid. All primers were ordered from Genewiz (Supplementary Table 2). The presence of the intended KaiC_RS mutations in the plasmid was confirmed by DNA sequencing by Genewiz using primers ordered from the same company (listed in Supplementary Table 2).

Both KaiC_SE and KaiA_SE plasmids were a gift from E. K. O’Shea. Expression and purification were performed according to a previously described procedure⁶.

Expression and purification of KaiC_RS and KaiB_RS from R.
sphaeroides

KaiC_RS, KaiC_RS mutants and KaiB_RS were expressed in Escherichia coli BL21(DE3) cells (New England Biolabs) harbouring the plasmid pETM-41 containing the kaiC_RS or kaiB_RS gene. Three colonies from a freshly prepared transformation plate were inoculated into 1 litre of TB medium containing 50 μg ml^–1 kanamycin. This culture was grown at 25 °C with shaking at 220 r.p.m. for 48 h without IPTG induction (leaky expression). The cells were pelleted by centrifugation at 4,200 r.p.m. for 15 min at 4 °C and stored at −80 °C.

Frozen cell pellets of KaiC_RS and KaiC_RS mutants were resuspended into lysate buffer (buffer A_C-RS) containing 1× EDTA-free protease inhibitor cocktail (Thermo Fisher Scientific), DNAse I (Sigma Aldrich) and lysozyme (Sigma Aldrich), and the lysate was sonicated for 10–15 min (20 s on, 30 s off, output power less than 40%) on ice followed by centrifugation at 18,000 r.p.m. at 4 °C for 45 min to remove cell debris. The lysate was filtered through a 0.45 μm filter and then loaded on HisTrap HP prepacked Ni-sepharose columns (Cytiva) pre-equilibrated with buffer A_C-RS at 0.5 ml min^–1. The column was washed with buffer A_C-RS at 1 ml min^–1 until the UV absorbance returned to baseline. Impurities were then washed with 15% buffer B_C-RS, and the protein was eluted with 50% buffer B_C-RS. The eluted complex was diluted with 1.5-fold dialysis buffer_C-RS then subjected to in-house prepared His-tagged TEV protease (1:10, TEVP:KaiC_RS molar ratio) cleavage to remove the His₆–MBP tag from KaiC_RS (wild-type and mutants) overnight at 4 °C in 6–8 kDa snakeskin dialysis tubing (Thermo Fisher Scientific) that was exchanged against dialysis buffer_C-RS. Cleaved KaiC_RS was filtered through a 1 μm filter and once again loaded onto HisTrap HP prepacked Ni-sepharose columns at 0.5 ml min^–1 to remove His-tagged TEV protease, His₆–MBP tag and uncleaved protein. The flow through was concentrated using a Millipore Amicon Ultra-15 centrifugal filter device (10 kDa cut-off) and immediately passed through a HiPrep Sephacryl S-400 HR column (Cytiva) pre-equilibrated with buffer C_C-RS. Protein was purified to homogeneity with a single band on Bis-Tris 4–12% gradient SDS–PAGE gel (Genscript) at 62.5 kDa. All protein purification steps were done at 4 °C or on ice. Protein was aliquoted and flash-frozen before storage at −80 °C until further use. The protein concentration was measured using a Microplate BCA Protein Assay kit (Thermo Fisher Scientific) on a SpectraMax MiniMax 300 imaging cytometer using BSA as a standard curve. Typical yields of KaiC_RS (wild-type and mutants) were 20–40 mg l^–1 of culture.

To test whether the purified KaiC_RS had any ATPase contamination, Q-sepharose HP columns (Cytiva) pre-equilibrated with buffer D_C-RS was used before the final HiPrep Sephacryl S-400 HR column. The protein was eluted with 5 CV of a linear gradient from 0 to 100% buffer E_C-RS. The ATPase activity of the protein samples purified using Q-sepharose HP columns was identical to the samples without this additional purification step. Buffer A_C-RS comprised 50 mM Tris-base (pH 7.5), 250 mM NaCl, 10 mM imidazole, 2 mM TCEP, 5 mM MgCl₂, 1 mM ATP and 10% glycerol (v/v). Buffer B_C-RS comprised 50 mM Tris-base (pH 7.5), 250 mM NaCl, 500 mM imidazole, 2 mM TCEP, 5 mM MgCl₂, 1 mM ATP and 10% glycerol (v/v). Dialysis_C-RS comprised 50 mM Tris-base (pH 7.0), 50 mM NaCl, 2 mM TCEP, 5 mM MgCl₂, 1 mM ATP and 10% glycerol (v/v). Buffer C_C-RS comprised 50 mM Tris-base (pH 7.0), 50 mM NaCl, 2 mM TCEP, 5 mM MgCl₂, 1 mM ATP and 10% glycerol (v/v). Buffer D_C-RS comprised50 mM Tris-base (pH 7.0), 2 mM TCEP, 5 mM MgCl₂, 1 mM ATP and 10% glycerol (v/v). Buffer E_C-RS comprised 50 mM Tris-base (pH 7.0), 1 M NaCl, 2 mM TCEP, 5 mM MgCl₂, 1 mM ATP and 10% glycerol (v/v).

The purification of KaiB_RS was similar to KaiC_RS, but with slight modifications as outlined below. After sonication and centrifugation to remove cell debris, the lysate was filtered through a 0.22 μm filter and then passed through HisTrap HP prepacked Ni Sepharose columns, pre-equilibrated with buffer A_B-RS. The column was washed with buffer A_B-RS until the UV absorbance returned to baseline. Impurities were washed with 5% buffer B_B-RS, and the protein was eluted with 50% buffer B_B-RS. The fusion protein was concentrated down to around 30 ml using Amicon stirred cells (Millipore Sigma) with 10 kDa cut-off. In-house prepared His-tagged TEV protease was added, and the fusion protein was cleaved overnight at 4 °C in a 3.5 kDa dialysis cassette that was exchanged against dialysis_B-RS buffer. The cleaved KaiB_RS was passed through HisTrap HP prepacked Ni-sepharose columns and concentrated down to about 10 ml using a Millipore Amicon Ultra-15 centrifugal filter device (3.5 kDa cut-off). The protein sample was then loaded onto a 26/60 Superdex S75 gel-filtration column (Cytiva) pre-equilibrated with buffer C_B-RS at 4 °C. The eluted protein was loaded onto Q-sepharose HP columns pre-equilibrated with buffer D_B-RS to remove ATPase contamination. The protein was eluted out in the flow through. A gradient from 0 to 100% buffer E_B-RS was passed through Q-sepharose HP columns to ensure that no KaiB_RS was bound to the columns. Protein was purified to homogeneity with the single band on Bis-Tris 4–12% gradient SDS–PAGE gels at 10.3 kDa. Protein was aliquoted and flash-frozen before storage at −80 °C until use. The protein concentration was measured using a Microplate BCA Protein Assay kit (Thermo Fisher Scientific) on a SpectraMax MiniMax 300 imaging cytometer using BSA as a standard curve. Typical yields of KaiB_RS were 30–40 mg l^–1 of culture.

Buffer A_B-RS comprised 50 mM Tris-base (pH 7.5), 250 mM NaCl, 10 mM imidazole, 2 mM TCEP and 10% glycerol (v/v). Buffer B_B-RS comprised 50 mM Tris-base (pH 7.5), 250 mM NaCl, 500 mM imidazole, 2 mM TCEP and 10% glycerol (v/v). Dialysis_B-RS comprised 50 mM Tris-base (pH 7.5), 250 mM NaCl, 2 mM TCEP and 10% glycerol (v/v). Buffer C_B-RS comprised 50 mM Tris-base (pH 7.5), 50 mM NaCl, 2 mM TCEP and 10% glycerol (v/v). Buffer D_B-RS comprised 50 mM Tris-base (pH 7.5), 2 mM TCEP and 10% glycerol (v/v). Buffer E_B-RS comprised 50 mM Tris-base (pH 7.5), 1 M NaCl, 2 mM TCEP and 10% glycerol (v/v).

Phylogenetic tree of KaiC

Protein sequences used in this study were identified in a multistep process. In the first step, a selection of sequences was identified using the BLASTP algorithm, utilizing a query based on the protein sequence for KaiC from S. elongatus (GenBank: WP_011242648.1)³⁸. The query was run against NCBI’s non-redundant protein database with the exclusion of models or uncultured and environmental sample sequences. A multiple sequence alignment of the selected 1,538 sequences was generated using MAFFT^39,40,41 (Supplementary Dataset 1). This alignment was used as input to generate an initial phylogenetic tree for KaiC with RAxML (v.8.2.9)⁴² using the PROTGAMMALG model. The generated tree was then used to identify the emergence of KaiB to create the final tree that focused on systems containing either KaiBC or KaiABC. To do so, a BLASTP search was performed for each branch tip with the KaiB sequence from S. elongatus, with the results restricted to the organism at which the branch tip was identified from. The observed branch point of emergence of KaiB agrees well with previous results in which it was shown that KaiB is mainly seen in non-archaea, non-proteobacteria².

To ensure the best possible sequence coverage, a BLASTP search using KaiB as query (GenBank: WP_011242647.1) was performed. The resulting sequences were then used to identify the organisms that they came from, which allowed us to create a list of organisms with an identified KaiB sequence. This list was then used to select for a subsequent BLASTP search using KaiC as query and therefore to identify only KaiC sequences for organisms that contain both KaiB and KaiC. A spot check was run to confirm that, for example, KaiA was indeed found among all cyanobacteria identified except for Prochlorococcus marinus. The obtained sequences were trimmed down to only include sequences with a sequence homology of 90% or less using CD-HIT⁴³ to arrive at a total of 401 sequences. For the calculation of the phylogenetic tree, RecA from S. elongates was added to serve as the outgroup. Sequences were aligned using MAFFT^39,40,41 (Supplementary Dataset 2) with the E-INS-I algorithm⁴⁴. The multiple sequence alignment was then used as input for the phylogenetic tree calculation with IQ-TREE (v.1.6.beta5), using the LG-substitution matrix⁴⁵ with the freeRate model (using 10 categories; LG+R10)^46,47. To enable determination of branch support, an aBayes test⁴⁸, a SH-aLRT test (20,000 bootstrap replicates⁴⁹) and an ultrafast bootstrap (20,000 bootstrap replicates⁵⁰) were performed (Supplementary Dataset 1; branch supports in order: SH-aLRT support (%)/aBayes support/ultrafast bootstrap support (%)).

X-ray crystallography

KaiC_RS and KaiC_RS-Δcoil crystals were obtained by sitting-drop vapour diffusion using a 96-well Intelli-Plate (102-0001-00, Art Robbins) at 291 K. Drops contained 0.5 μl crystallization solution, 0.5 μl protein at 10 mg ml^–1 in 20 mM MOPS pH 6.5, 50 mM NaCl, 2 mM TCEP, 5 mM MgCl₂, 3 mM ATP and 1 mM AMPPCP, and were equilibrated against 50 μl of the solution in the reservoir. The KaiC_RS crystallization solution consisted of 200 mM magnesium chloride hexahydrate, 100 mM HEPES pH 7.5 and 30% (w/v) PEG 400. KaiC_RS-Δcoil crystals were grown using 200 mM ammonium acetate, 100 mM sodium citrate tribasic dihydrate pH 5.6 and 30% (w/v) PEG 4,000.

The PEG 400 in the KaiC_RS crystallization solution acted as a cryoprotectant, whereas KaiC_RS-Δcoil crystals were cryoprotected in LV Cryo Oil (MiTeGen). Single crystals were cooled in liquid nitrogen, and X-ray diffraction images were collected at ALS beamline 8.2.1 at 100 K (data collection details are described in Extended Data Table 1). The data were indexed and integrated in iMosflm⁵¹, and scaled and merged in Aimless⁵².

To obtain a structural model of KaiC_RS, first the KaiC_RS-Δcoil structure was solved by molecular replacement in MRage⁵³ using the KaiC_RS sequence (residues 1–490) as input to search for homologues in the PDB database. The initial KaiC_RS-Δcoil structure based on the KaiC_SE (PDB: 1TF7 (ref. ³⁶)) was manually rebuilt in Coot (v.0.9.81)⁵⁴ and refined in Phenix (v.1.20.1-4487)⁵⁵. Finally, the KaiC_RS-Δcoil structure was used as the molecular replacement search model in Phaser⁵⁶ to solve the full-length KaiC_RS structure.

The assigned space groups were validated in Zanuda⁵⁷, and the position of the asymmetric unit in the unit cell was standardized using Achesym⁵⁸. KaiC_RS coiled-coil registers were analysed using SamCC-Turbo (v.0.0.2) with the default socket cut-off value of 7.4 (ref. ⁵⁹). The images of protein structures were rendered using PyMOL (v.2.6.0)⁶⁰.

Tunnel detection and calculation were performed using CAVER 3.0.2 PyMOL plugin⁶¹, with the minimum probe radius varying between 0.9 and 1.1. Default values were used for all other parameters. All atoms except waters were used in the calculation. The residue selection for starting point consisted of Glu62, Glu63 and ADP602. The catalytic position of the water in the CI domain was modelled from the crystal structure of the transition-state analogue-bound F₁-ATPase (PDB: 1w0j, water 2,064 from chain D⁶²).

Cryo-EM and image processing

For preparation of EM grids, 3–4 μl of 4.3 mg ml^–1 (per monomer concentration) of sample in 20 mM MOPS pH 6.50, 50 mM NaCl, 2 mM TCEP, 10 mM MgCl₂ and 2 mM ATP was applied to glow-discharged 1.2/1.3 400 mesh C-flat carbon-coated copper grids (Protochips). The grids were frozen using a Vitrobot Mark IV (ThermoFisher) at 4 °C and 95% humidity, with a blot time of 4 s. All datasets were collected on a Titan Krios operated at an acceleration voltage of 300 keV, with a GIF quantum energy filter (Gatan) and a GATAN K2 Summit direct electron detector controlled by SerialEM⁶³.

Inspection of the raw cryo-EM images revealed some heterogeneity in the relative orientations between individual hexamers of the dodecameric particles, presumably due to inherent flexibility in the coiled-coil regions, which limited the resolution to 3.3–3.4 Å. To obtain higher resolution reconstructions, the dodecamers were split and processed as individual hexamers, with C6 symmetry being applied throughout processing. To reconstitute the full dodecamer reconstruction, two copies of the hexamer reconstruction were overlaid on top of each other using the ‘fit in map’ function in Chimera⁶⁴ to fit one hexamer into the lower resolution end density of the other. The overlaid hexamers were then combined, creating a new map in which each voxel takes the value from the hexamer with highest absolute value.

For KaiC_RS-S413E/S414E alone, a dataset of approximately 2,500 movies was collected. The movies were recorded with a pixel size of 1.074 Å, including 70 frames and with an exposure rate of 1.31 e⁻ per Ų per frame. Approximately 825,000 particles were picked, and after 2D classification, around 320,000 particles from good class averages were carried forward for further processing. The final measured resolution of the reconstruction was 2.9 Å (Extended Data Fig. 4a).

For the KaiC_RS-S413E/S414E:KaiB_RS complex, a dataset of around 2,000 movies was collected. The movies were recorded with a pixel size of 1.023 Å, including 70 frames and with an exposure rate of 1.35 e⁻ per Ų per frame. About 440,000 particles were picked, and after 2D classification around 190,000 particles from good class averages were carried forward for further processing. The final measured resolution of the reconstruction was 2.7 Å (Extended Data Fig. 4b).

All data processing was carried out using cisTEM (v.2.0.0)⁶⁵, and followed the workflow of motion correction, CTF parameter estimation, particle picking, 2D classification, ab initio 3D map generation, 3D refinement, 3D classification, per-particle CTF refinement and B-factor sharpening. The highest resolution of 3D refinement used was 4 Å for both reconstructions, and final resolutions were estimated using the cisTEM PartFSC and a threshold of 0.143.

To validate the combined dodecamer structure, we also processed both datasets as full D6 symmetric dodecamers (Extended Data Fig. 4c,d). This was accomplished by extracting the picked hexamers into a large box size and performing 2D classification with automatic centring. Clear dodecamer class averages were then selected and re-extracted from the original images, with picking coordinates that were adjusted by the translation required to match the centred class average. After this centring, duplicate picks were removed to obtain the final dodecamer particle stacks. These stacks were processed as described above, with the highest resolution of 3D refinement used as 4.25 Å.

In an attempt to find deviations from D6 symmetry, we also calculated reconstructions for both structures assuming C1 symmetry, starting from the ab initio 3D step. The resulting refined C1 structures did not exhibit detectable departures from D6 symmetry (Extended Data Fig. 4e). We therefore present symmetrized volumes as our final result.

The cryo-EM structures were built using the KaiC_RS model obtained by X-ray crystallography and fold-switch-stabilized KaiB_TE (PDB: 5JWO (ref. ²⁶)) as starting points. The models were constructed using Coot (v.0.9.81)⁵⁴, and refinement was carried out using Phenix (v.1.20.1-4487)⁵⁵.

Preparation of unphosphorylated KaiC_RS

Purified KaiC_RS (about 20 μM) from −80 °C was dialysed in 20 mM MOPS (pH 6.5), 50 mM NaCl, 2 mM TCEP, 10 mM MgCl₂ and 0.1 mM ADP overnight at 4 °C to remove glycerol and to replace ATP with ADP. The dialysed KaiC_RS was then heated at 30 °C for 4 h to obtain fully unphosphorylated KaiC_RS bound with ADP, and the sample was then passed through 0.22 μm Spin-X centrifuge tube filters (Corning). The sample was concentrated to a higher concentration (less than 100 μM) at 4 °C. The protein concentration was measured using a BCA assay.

In vitro KaiBC_RS reaction

Kinetics of KaiC_RS autophosphorylation in the presence and absence of KaiB_RS

Unphosphorylated KaiC_RS (3.5 μM, prepared as described above) in the presence or absence of KaiB_RS (3.5 μM and 35 μM) was preincubated at 20, 25, 30 and 35 °C for 1 h in 20 mM MOPS (pH 6.5), 50 mM NaCl, 2 mM TCEP, 10 mM MgCl₂ and 0.1 mM ADP. The reactions were started by adding 3.9 mM ATP to obtain a final concentration of 4 mM nucleotide in the presence of 2 U ml^–1 pyruvate kinase (Millipore Sigma) and 10 mM phosphoenolpyruvate (Millipore Sigma) to regenerate ATP during the reaction. The reaction samples were sampled by hand at specific time points and mixed with an equal amount of loading dye (stock concentration of 0.1 M Tris-base (pH 7.5), 4% SDS, 0.2% bromophenol blue, 30% glycerol and 0.5 M 2-mercaptoethanol). The mixed samples were then stored at −20 °C until further use.

Kinetics of KaiC_RS autodephosphorylation in the presence and absence of KaiB_RS

Purified KaiC_RS (around 20 μM) was dialysed in reaction buffer containing 20 mM MOPS (pH 6.5), 50 mM NaCl, 2 mM TCEP, 10 mM MgCl₂ and 0.1 mM ADP overnight at 4 °C to remove glycerol and to generate KaiC_RS bound with ADP. After dialysis at 4 °C, KaiC_RS exists in two states: 50% unphosphorylated and 50% single phosphorylated at Ser413 (pSer413), which were confirmed by tandem mass spectrometry (data not shown). The autodephosphorylation reaction was started by adding KaiC_RS or KaiC_RS in the presence of KaiB_RS (3.5 μM) into reaction buffer pre-equilibrated at 30 °C.

Oscillation of KaiBC_RS

Dialysed KaiC_RS (3.5 μM) was preincubated at 35 °C for 30 min in 20 mM MOPS (pH 6.5), 50 mM NaCl, 2 mM TCEP, 10 mM MgCl₂ and 0.1 mM ADP in the presence or absence of KaiB_RS (3.5 μM). The reactions were started by adding 4 mM ATP and reaction samples were collected at specific time points for 10% SDS–PAGE and HPLC analysis to identify phosphorylation state of KaiC_RS and amount of nucleotide at each time point, respectively.

Controlling ATP-to-ADP ratio to mimic daytime and night time

KaiC_RS was dialysed in reaction buffer containing 20 mM MOPS (pH 6.5), 50 mM NaCl, 2 mM TCEP, 10 mM MgCl₂ and 1 mM ATP overnight at 4 °C. To start the reaction as shown in Fig. 3c, KaiC_RS (3.5 μM) in the absence or presence of KaiB_RS (3.5 μM) was mixed with additional ATP (final 4 mM to mimic daytime), and the reaction samples (500 μl) were added into a D-Tube Dialyzer (midi 3.5 kDa cut-off, EMD Millipore) that was exchanged against 4 mM ATP buffer (400 ml). After the 12-h time point, the reaction samples were transferred into preincubated 25% ATP/ADP buffer (400 ml) that mimics the night time. After the 24-h time point, the same samples were changed into preincubated 4 mM ATP to mimic the daytime again.

To start the experiment as shown in Extended Data Fig. 6a, KaiC_RS (35 μM) in the presence of 3 mM ATP in 20 mM MOPS (pH 6.5), 50 mM NaCl, 2 mM TCEP and 10 mM MgCl₂ was heated at 35 °C for 25 min to generate fully phosphorylated KaiC_RS. The KaiC_RS sample was then diluted 10-fold into 25% ATP/ADP buffer pre-equilibrated at 30 °C to final concentration of KaiC_RS (3.5 μM) and KaiB_RS (3.5 μM or 35 μM). The reaction samples (300 μl) were added into a D-Tube Dialyzer (midi 3.5 kDa cut-off, EMD Millipore) that was exchanged against 25% ATP/ADP buffer (300 ml).

During the reaction, the samples were gently shaken in a 30 °C incubator, and reaction samples were collected at specific time points for 10% SDS–PAGE and HPLC analysis to identify the phosphorylation state of KaiC_RS and the amount of nucleotide at each time point, respectively.

The rationale for the ATP-to-ADP ratio at daytime and night time comes from two earlier literature reports. The change in ATP-to-ADP ratio at daytime and night time were directly measured in vivo in the strain R. sphaeroides²⁷, in which ATP is 2.0–2.4 mM during day and drops to 0.5–0.6 during night, and it is well known that the total nucleotide concentration stays constant. We chose the total nucleotide concentration of 4 mM in our in vitro work to be identical to the described in vitro experiments performed for the canonical KaiC_SE. Because of photosynthesis in daylight, virtually all nucleotide is ATP³³. We note that a slightly higher amount of ATP will not affect our results, as the affinity of KaiC_RS for ATP is higher than for ADP.

Separation of unphosphorylated, single and double phosphorylated KaiC_RS by SDS–PAGE

Unphosphorylated, single phosphorylated and double phosphorylated KaiC_RS were separated by 10% SDS–PAGE with 37.5:1 acrylamide:bis-acrylamide (Bio-Rad), 18 cm × 16 cm × 1 mm Tris-HCl gel with 1× Tris-glycine SDS running buffer (Invitrogen). The samples were heated at 95 °C for 3 min, and 400 ng of material was loaded onto the Tris-HCl gel. The gel was run with a constant current of 35 mA, 150 W, and the voltage was greater than 700 V for 5.5 h in a cold room, with a water bath set to 12 °C using a Hoefer SE600 electrophoresis unit.

Unphosphorylated and phosphorylated KaiC_RS-Δcoil were separated by Zn²⁺ Phos-tag SDS–PAGE with 10% acrylamide gel containing 50 μM Phos-tag acrylamide (Wako). The gel was run with a constant current of 30 mA for 5 h 30 min in a cold-room, with 1 μg per well protein samples pre-heated at 95 °C for 3 min.

The gels were stained overnight at room temperature with InstantBlue protein gel stain (Expedeon) with gentle shaking and destained with distilled water until bands were clearly visible. The gels were imaged on a ChemiDoc Imager (Bio-Rad), and Image Lab software (Bio-Rad) was used for analysis.

Statistics and reproducibility for gel electrophoresis

Data shown in main text figures and Extended Data figures are representative SDS–PAGE gels for at least three independent biological replicates (n = 3), except for experiments presented in Fig. 3a,c, which were performed in duplicate.

Oligomerization state of KaiC_RS and KaiB_RS

Gel-filtration chromatography

KaiC_RS, unphosphorylated KaiC_RS and all KaiC_RS mutants with a concentration of around 40–80 μM were loaded with a flow rate of 0.2 ml min^–1 onto a prepacked Superdex-200 10/300 GL (GE Healthcare) pre-equilibrated with 20 mM MOPS (pH 6.5), 50 mM NaCl, 2 mM TCEP, 10 mM MgCl₂ and 1 mM ATP (0.1 mM ADP for unphosphorylated KaiC_RS) at 4 °C using an ÄKTA Pure system (GE Healthcare). KaiB_RS (0.5 mM) was loaded onto Superdex-75 10/300 GL (GE Healthcare) pre-equilibrated with 20 mM MOPS (pH 6.5), 50 mM NaCl and 2 mM TCEP at 4 °C. The eluate was collected in fractions of 1 ml each and subjected to SDS–PAGE analysis. A standard curve (that is, molecular weight versus elution time) was determined for the column using molecular weight protein standards (Bio-Rad) run in the same buffer and flow rate. The protein standard mixture contained thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B₁₂ (1.35 kDa).

Analytical ultracentrifugation

Sedimentation velocity centrifugation experiments were run at 50,000 r.p.m. (for KaiB_RS) and 30,000 r.p.m. (for KaiC_RS wild-type and mutants and KaiC_SE), with continuous scans from 5.8 to 7.3 cm at 0.005 cm intervals at 20 °C on a Beckman Optima XL-A (Beckman-Coulter) equipped with absorption optics and a four-hole An60Ti rotor. Measurements were set up at 280 nm (for KaiB_RS) and 295 nm (for KaiC_RS and KaiC_SE) to avoid interference from ATP. The software package SEDFIT (v.14.1) was used for data evaluation^66. KaiC_RS and KaiC_RS-Δcoil (100 μM) were prepared in 20 mM MOPS (pH 6.5), 50 mM NaCl, 2 mM TCEP, 10 mM MgCl₂ and 1 mM ATP. KaiC_SE (100 μM) was prepared in 20 mM MOPS (pH 8.0), 150 mM NaCl, 2 mM TCEP, 5 mM MgCl₂, and 1 mM ATP. KaiB_RS (500 μM) was prepared in 20 mM MOPS (pH 6.5), 50 mM NaCl and 2 mM TCEP.

ATPase activity

Purified KaiC_RS (both wild-type and mutant forms (around 20 μM)) and KaiB_RS (about 90 μM) were dialysed in 20 mM MOPS (pH 6.5), 50 mM NaCl, 2 mM TCEP, 10 mM MgCl₂ and 1 mM ATP (reaction buffer) overnight at 4 °C. The samples were passed through 0.22 μm Spin-X centrifuge tube filters, and concentrations were measured using a BCA assay before setting up the reactions. Typical KaiC_RS or KaiBC_RS reactions contained 3.5 μM KaiC_RS (wild type and mutants) and 3.5 μM KaiB_RS in reaction buffer with a final concentration of 4 mM ATP. The samples were incubated at the indicated temperatures and were sampled by hand at specific time points. Next 10 μl of sample was quenched with 10 μl of 10% trichloroacetic acid (Millipore Sigma), and the mixture was passed through a 0.22 μm Spin-X centrifuge tube filter to remove the precipitated protein. The flow through sample was then re-adjusted to pH 6.2 for nucleotide separation by adding 10 μl of 0.75 M HEPES, pH 8.0. The final samples were kept at −20 °C until HPLC analysis.

Three microlitres of each sample were injected with a high-precision autosampler (injection error of <0.1 μl, resulting in a maximum systemic error of about 6%) to a reverse-phase HPLC instrument with an ACE 5 μm particle size, C18-AR and 100 Å pore size column (Advanced Chromatography Technologies). The instrument was pre-equilibrated with 100 mM potassium phosphate pH 6.2 with a flow rate of 0.4 ml min^–1. Using pure nucleotide samples, the retention times of ATP, ADP and AMP were determined to be 2.6, 3.1 and 4.4 min, respectively. The concentration of each nucleotide was calculated from the relative ratio of the peak areas and the total nucleotide concentration. To determine ATPase activity rates, the observed rate constants were determined from at least five data points for each temperature using initial rate analysis and least-squares linear regression (Extended Data Figs. 6c,d and 8b–d). The mean values and uncertainties (s.d.) shown in Fig. 3d, Extended Data Figs. 6 and 8 were derived from three replicate experiments. KaleidaGraph (v.4.5.3; Synergy) was used for data analysis and plotting.

Nucleotide exchange

KaiC_RS (wild type or mutants) and KaiB_RS were dialysed into 20 mM MOPS (pH 6.5), 50 mM NaCl, 2 mM TCEP, 10 mM MgCl₂ and 50 μM ATP overnight at 4 °C. The samples were passed through 0.22 μm Spin-X centrifuge tube filters, and the protein concentration was measured using a BCA assay. The reaction contained 3.5 μM of KaiC_RS-S413E and/or 35 μM of KaiB_RS, and samples were incubated at 20, 25, 30 and 35 °C for 16–24 h in the presence of an ATP-recycling system. The reactions were started by adding 250 μM of mant-ATP (Jena Bioscience). The spectrum was measured using the fluorescence energy transfer from tryptophan residues in KaiC_RS to mant-ATP by exciting the sample at 290 nm (2.5 nm bandwidth) and collecting the emission intensity from 320 nm to 550 nm (5 nm bandwidth) in increments of 2 nm. To measure the nucleotide exchange rate, the maximum change in fluorescence intensity at 440 nm (ΔF_440 nm) was followed for a total time of 1,800 s in 15 s increments with anti-photobleaching mode on FluoroMax-4 spectrofluorometer (Horiba Scientific) equipped with a water bath to control the temperature. There are two tryptophan residues within 5 Å from the nucleotide-binding site in the KaiCII_RS domain and no tryptophan residue close to the nucleotide-binding site in the KaiCI_RS domain, so the nucleotide exchange observed in the experiments are for the KaiCII_RS domain. To ensure that the exchange rate observed in the experiments are from nucleotide exchange in the KaiCII_RS domain, KaiCI_RS (which only contains the CI domain) was tested; no change in fluorescence was observed following the addition of mant-ATP (Extended Data Fig. 8h).

The experiments for KaiC_SE alone and with KaiC_SE mixed in and for KaiA_SE were performed in a similar way, except that KaiC_SE and KaiA_SE were dialysed in 20 mM MOPS (pH 8.0), 150 mM NaCl, 2mM TCEP, 10 mM MgCl₂ and 50 μM ATP overnight at 4 °C. KaiA_SE was incubated with KaiC_SE for 1 h at 30 °C before adding 250 μM mant-ATP.

For the nucleotide preference experiment, KaiC_RS-S413E/S414E and KaiC_RS-S413A/S414A were dialysed in 20 mM MOPS (pH 6.5), 50 mM NaCl, 2 mM TCEP, 10 mM MgCl₂ and 20 μM ADP overnight at 4 °C. KaiC_RS-S413E/S414E or KaiC_RS-S413A/S414A (3.5 μM) was first mixed with mant-ATPγS or mant-ADP (150 μM), and the kinetic trace at 440 nm was recorded at 30 °C. After the fluorescence trace at 440 nm reached a plateau, which indicates that the nucleotide analogue was fully bound to the protein, a 27-fold excess of ATP (4 mM) was added to displace the bound nucleotide analogue, and the decay of fluorescence intensity was recorded at 440 nm at 30 °C. The experiments were run in triplicate, and results were averaged and fitted to a single exponential decay.

Analysis was performed by fitting individual traces to an exponential equation using KinTek Explorer software^67,68], and error bars denote the standard errors as obtained from triplicate experiments. KaleidaGraph (v.4.5.3; Synergy) was used for data plotting.

³²P-ATP radioactive labelling and experiment

³²P-labelled KaiC_RS was prepared by mixing unphosphorylated KaiC_RS (10 μM) with 0.46 μM [γ-³²P]ATP (3,000 Ci mmol^–1, PerkinElmer) and 500 μM ATP in 20 mM MOPS (pH 6.5), 50 mM NaCl, 2 mM TCEP and 10 mM MgCl₂ at 35 °C for 30 min and then immediately switched to 4 °C to prevent dephosphorylation of KaiC_RS. The ³²P-labelled KaiC_RS sample was passed through Zeba spin desalting columns (Thermo Fisher Scientific) pre-equilibrated with 20 mM MOPS (pH 6.5), 50 mM NaCl, 2 mM TCEP and 10 mM MgCl₂ twice at 4 °C. The sample was incubated with buffer containing 1 mM ADP overnight at 4 °C to obtain ³²P-labelled KaiC_RS bound with ADP. The sample was passed through a final Zeba desalting column pre-equilibrated with 20 mM MOPS (pH 6.5), 50 mM NaCl, 2 mM TCEP and 10 mM MgCl₂ at 4 °C, and the solution was then incubated with 8 mM ADP in the presence or absence of KaiB_RS at 4 °C for 1 h. The samples were then diluted in 20 mM MOPS (pH 6.5), 50 mM NaCl, 2 mM TCEP and 10 mM MgCl₂ preincubated at 30 °C to obtain a final concentration of ³²P-labelled KaiC_RS (5 μM), KaiB_RS (20 μM) and ADP (4 mM). The reactions were incubated at 30 °C, and at different time points, aliquots were taken (1.5 μl). The reactions were stopped by adding 1.5 μl Laemmli sample buffer (62.5 mM Tris (pH 6.8), 2% SDS, 25% glycerol and 0.01% bromophenol blue) supplemented with 5% (v/v) 2-mercaptoethanol.

The samples were spotted onto a TLC plate (PEI-cellulose F plates, Merck) and quickly dried with a blow-dryer for 30 s. The TLC plates were run first with distilled water as a mobile phase. After TLC plates were completely dried, 0.75 M KH₂PO₄ was used as the mobile phase to separate ³²P-labelled KaiC_RS, [γ-³²P]ATP and inorganic phosphate (³²P), as previously shown²⁹. The phosphor-screens were scanned on an Amersham Typhoon (GE Healthcare) at a resolution of 100 μm. ImageQuant TL 7.0 software was used for analysis.

Fluorescence anisotropy competition

Fluorescence anisotropy competition experiments were carried out using a FluoroMax-4 spectrofluorometer (Horiba Scientific) at 30 °C. Excitation and emission wavelengths for KaiB_RS labelled with 6-iodoacetamidofluorescein (6-IAF, Thermo Fisher Scientific) at Cys29 were set at 492 nm (5 nm bandwidth) and 520 nm (5 nm bandwidth), respectively, with fixed G factor (G factor of KaiB_RS–6IAF alone) to eliminate instrumental bias. The average anisotropy and standard error were calculated from ten replicate measurements.

KaiB_RS–6IAF was prepared by mixing degassed KaiB_RS (100 μM) with 20-fold excess of 6-IAF (stock 10 mM in 50% DMSO) in 20 mM Tris-base (pH 7.0), 50 mM NaCl and 1 mM TCEP (degassed). The reaction was incubated at room temperature for 4 h and dialysed against 20 mM Tris-base (pH 7.0), 50 mM NaCl and 1 mM TCEP in a 3.5 kDa dialysis cassette overnight at 4 °C to remove unreacted 6-IAF and small amounts of DMSO. The crosslinked sample was passed through a 0.22 μm Spin-X centrifuge tube filter and loaded onto Superdex-75 10/300 GL pre-equilibrated with 20 mM MOPS (pH 7.0), 50 mM NaCl and 1 mM TCEP with a 0.2 ml min^–1 flow rate to remove leftover unreacted 6-IAF. The sample was aliquoted and flash-frozen in liquid nitrogen and stored at −80 °C until use. All the crosslinked reactions were performed in the dark.

For the fluorescence anisotropy competitive binding experiment, KaiB_RS–6IAF (0.2–0.4 μM) was first incubated with wild-type or mutant KaiC_RS (1 μM, 60% increase in anisotropy in comparison to KaiB_RS–6IAF alone) in 20 mM MOPS (pH 6.5), 50 mM NaCl, 2 mM TCEP and 10 mM MgCl₂ in the presence of 4 mM ADP or 4 mM ATP with an ATP-recycling system (2 U ml^–1 pyruvate kinase and 10 mM phosphoenolpyruvate), then unlabelled KaiB_RS (0–50 μM) was added to the samples. The samples were incubated at 30 °C for 4 h or 12 h before measurement.

The decrease in fluorescence anisotropy (FA) versus concentration of unlabelled KaiB_RS was fitted to equation (1) using the Levenberg–Marquardt nonlinear fitting algorithm included in KaleidaGraph (Synergy Software) to obtain the half-maximum inhibitory concentration (IC₅₀) value. The K_d value can then be calculated from the IC₅₀ value using equation (2) as previously described⁶⁹.

Pull-down assays

Pull-down assays probe the interaction between KaiC_RS (wild type and mutants) with KaiB_RS (His₆-MBP-TEV-KaiB_RS). The His₆-MBP-TEV-KaiB_RS was expressed and purified as described above but without the TEV protease cleavage step. Wild-type or mutant KaiC_RS (3.5 μM) was mixed with KaiB_RS-tag (3.5 μM) in 20 mM MOPS (pH 6.5), 50 mM NaCl, 2 mM TCEP and 10 mM MgCl₂ in the presence of 4 mM ADP or 4 mM ATP with an ATP-recycling system in a final volume of 400 μl. The samples were incubated at 25 °C for 4 h or 24 h before loading onto a 500 μl spin column with 200 μl (prepared from 400 μl of 50% slurry) Talon beads (Takara) pre-equilibrated with sample buffer. The samples were incubated with the Talon beads for 30 min with gentle shaking, after which the flow through was collected by gravity into 1 ml Eppendorf tubes. The beads were washed three times with 400 μl sample buffer by gravity, then the samples were eluted with 200 μl of 0.5 M imidazole buffer by centrifugation at 1,000g for 1 min. The protein mixture, flow through, wash and eluted samples were run on a Bis-Tris 4–12% gradient SDS–PAGE gel with a molecular weight marker. The gels were stained with Coomassie blue and were imaged on a ChemiDoc Imager (Bio-Rad). Image Lab Software (Bio-Rad) was used for analysis.

The following control experiments were performed: (1) fusion KaiB_RS protein in the absence of KaiC_RS; and (2) KaiC_RS in the absence of fusion KaiB_RS. All the fusion KaiB_RS proteins came out only in the elution buffer in the first control experiment, which indicated that fusion KaiB_RS binds to Talon beads and the amount of KaiB_RS used did not overload the column. All KaiC_RS protein came out in the flow through in the second control experiment, which indicated there is no specific binding between KaiC_RS and the Talon beads.

Thermofluor assay

KaiC_RS and SYPRO Orange (Thermo Fisher Scientific) were used at final concentration of 3 μM and 10×, respectively. The experiments were carried out in 20 mM MOPS (pH 6.5), 50 mM NaCl, 2 mM TCEP, 10 mM MgCl₂ and 4 mM ADP or ATP. The samples were prepared to a final volume of 20 μl in a MicroAmp Fast Optical 96-well reaction plate (Applied Biosystems Life Technologies), and the plate was sealed with Axygen UltraClear sealing film (Corning). The assay plate was run in a StepOne Real-Time PCR instrument (Applied Biosystems Life Technologies) with melt curve set up. The temperature was continuously increased from 25 °C to 95 °C by 0.3 °C every 15 s. The data were fit with nonlinear fitting in KaleidaGraph (Synergy Software) to a Boltzmann sigmoidal curve (equation (3)).

where Y is the fluorescence intensity at temperature T, T is the temperature in degrees Celsius, Bottom is the baseline fluorescence at low temperature, Top is the maximum fluorescence at the top of the truncated data, c is the slope or steepness of the curve, and T_m is the melting temperature of the protein.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.