Abstract
The AAA+ family member KaiC is the central pacemaker for circadian rhythms in the cyanobacterium Synechococcus elongatus. Composed of two hexameric rings of adenosine triphosphatase (ATPase) domains with tightly coupled activities, KaiC undergoes a cycle of autophosphorylation and autodephosphorylation on its C-terminal (CII) domain that restricts binding of clock proteins on its N-terminal (CI) domain to the evening. Here, we use cryogenic-electron microscopy to investigate how daytime and nighttime states of CII regulate KaiB binding on CI. We find that the CII hexamer is destabilized during the day but takes on a rigidified C2-symmetric state at night, concomitant with ring-ring compression. Residues at the CI-CII interface are required for phospho-dependent KaiB association, coupling ATPase activity on CI to cooperative KaiB recruitment. Together, these studies clarify a key step in the regulation of cyanobacterial circadian rhythms by KaiC phosphorylation.
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Data availability
EMDB and PDB depositions: Compressed conformation of nighttime KaiC (PDB 7S65, EMDB-24850), extended conformation of nighttime KaiC (PDB 7S66, EMDB-24851) and extended conformation of daytime state KaiC (PDB 7S67, EMDB-24852). All data are available in the main text or the supplementary information. Source data are provided with this paper.
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Acknowledgements
We thank J.C. Ducom at Scripps Research High Performance Computing for computational support, as well as B. Anderson at the Scripps Research Electron Microscopy Facility for microscopy support. Funding for this work was provided through National Institutes of Health grant nos. R01GM121507 and R35GM141849 to C.L.P., R01NS095892 and R21GM142196 to G.C.L., R35GM118290 to S.S.G., R01GM107521 to A.L. and F32GM130070 to D.E., as well as the National Science Foundation grant no. NSF-CREST: Center for Cellular and Biomolecular Machines at the University of California, Merced (grant no. NSF-HRD-1547848) to A.L.
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Conceptualization was done by J.A.S., C.R.S., G.C.L. and C.L.P. The investigation was carried out by J.A.S., C.R.S., A.G.C., A.M.F., D.E., C.R.S., D.C.E. and J.G.P. Funding was acquired by S.S.G., A.L., G.C.L. and C.L.P. Project administration was done by J.A.S., G.C.L. and C.L.P. The study was supervised by S.S.G., A.L., G.C.L. and C.L.P. The original draft was written by J.A.S., C.R.S., G.C.L. and C.L.P. Review and editing of the draft was done by J.A.S., C.R.S., S.S.G., A.L., G.C.L. and C.L.P.
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Extended data
Extended Data Fig. 1 Image processing pipeline and validation of C6 symmetric KaiC-AE structure.
a, Image processing pipeline for KaiC-EA datasets with fluorinated fos-choline-8. b, Local resolution estimation of cryo-EM reconstruction calculated by RELION52. c, Euler distribution plots depicting particle orientations present in final reconstruction. More populated views are colored in red while less populated are depicted in blue. d, 3D Fourier Shell Correlation (3DFSC)53 of final daytime state reconstruction, with a global resolution of 3.8 Å at FSC = 0.143.
Extended Data Fig. 2 CII nucleotide state in extended and compressed KaiC structures.
a, Electron volume and atomic model for CII nucleotide in the KaiC-AE structure determined in the presence of 4 mM fos-choline, b the C6-symmetric KaiC-EA structure and c extended or d compressed subunits of the C2-symmetric nighttime KaiC structure.
Extended Data Fig. 3 Image processing pipeline and validation of KaiC-EA.
a, Image processing pipeline for two combined KaiC-EA datasets: a 40° tilted dataset, and one collected on thin carbon. b, Local resolution estimation of cryo-EM reconstructions calculated by RELION52. c, Euler distribution plots depicting particle orientations present in final reconstructions. More populated views are colored in red. d, 3D Fourier Shell Correlation (3DFSC)53 of final nighttime state reconstructions, with a global resolution of 2.8 Å for the C6-state, and 3.2 Å for the C2-state at FSC = 0.143.
Extended Data Fig. 4 Structural comparison of C2-symmetric ATPase structures.
Seam protomers are depicted in blue while ATP and ADP nucleotides are shown in white and green, respectively. Apo nucleotide pockets are indicated with dashed circles. While other C2-symmetric ATPases are unliganded at their seam protomers, KaiC is ADP-bound at these subunits.
Extended Data Fig. 5 Allostery about the CII ring regulates KaiC autophosphorylation.
a, In the expanded state, the A-loop of an adjacent protomer (yellow) is positioned near the phosphosite-adjacent 422-loop through a hydrophobic interaction between I490 and M420. E444’s sidechain forms a hydrogen bond with the mainchain nitrogen of I490 in trans. In the compressed state of KaiC-EA, E444 in the ADP-bound protomers is positioned away from the pore and no longer stabilizes the A-loops, allowing them to become disordered and causing the 422-loops to collapse towards phosphosite−containing α9. Binding of KaiA to the C-terminus of KaiC may disrupt this tripartite interaction and stimulate hyperphosphorylation. b, Sypro orange fluorescence of KaiC mutants that were run on a 10% denaturing polyacrylamide gel containing 50 µM Phos-tagTM reagent and 100 µM Mn2+. c, Densitometric analysis of bands corresponding to phosphorylated and unphosphorylated KaiC. Gray bars represent mean ± standard deviation for n = 3 repeats.
Extended Data Fig. 6 KaiC residue Y402 exhibits rotameric heterogeneity in the compressed conformation.
a, Close-up view of the two Y402 rotamer conformations from the compressed protomer of the C2-symmetric KaiC-EA structure. Cryo-EM density is depicted in white and semi-transparent for clarity. Analysis from the modeling software Coot59 indicates that both rotamers are allowed with similar probabilities. Model vs. Data Cross Correlation in Phenix60 indicates that Rotamer 1 has a higher correlation compared to Rotamer 2, but that both are favorable. b, Superposition of compressed protomer B with expanded protomer A, aligned by the CII domain. The rotameric heterogeneity of Y402 in the compressed protomer is associated with rotameric deviations in nearby residues in comparison to an expanded protomer.
Extended Data Fig. 7 Multiple sequence alignment of key KaiC regions from various species of cyanobacteria.
a, Protein sequences of the CI and b CII regions of KaiC from eight distinct strains of cyanobacteria. Sequences are presented with each domain grouped to together and aligned amongst the species, and also aligned about the CI and CII domains within a given protein sequence. Key residues are highlighted with colors illustrating their conservation between the CI and CII domains.
Extended Data Fig. 8 The arginine tetrad creates a network of CI-CI trans interactions.
a, The 4TL8 structure40 is shown as light blue ribbons with the P-loops displayed in darker blue. Arginine tetrad side chains at clockwise (b) or counterclockwise (c) subunits as well as their electrostatic interaction partners are shown in brown. Average interatomic distance from the six interfaces are shown, as tabulated in Supplementary Table 3.
Extended Data Fig. 9 Cooperativity analysis of KaiC mutants.
a, Thermodynamic model, based on the one originally derived for heterotropic cooperativity in Chavan et al.34. In this case, the heterotropic cooperativity factor, S, is replaced with the homotropic factor represented by unlabeled KaiB-I87A (fsKaiB), which binds much tighter to KaiC than wild-type KaiB10. Wild-type KaiB is monitored, as indicated by an orange asterisk. b, 2-dimensional titrations are overlaid with titration curves derived from least-squares fitting of the data from each individual dataset. For unsubstituted (WT) KaiC-EA, the 95% confidence intervals from n = 1,000 Monte Carlo simulation is reported. These represent the n = 25 and n = 975 values from the simulation, which gave median (n = 500) values of 22, 16 and 25 (top panel to bottom panel). For each 2D-titration of the mutant nighttime KaiC variants, best fit cooperativity indices from least-squares analysis are reported as best fit ± standard error. See methods for more information on how these values and standard errors were determined.
Extended Data Fig. 10 Analysis of CI-CI nucleotide contacts from PDB 4TLA.
a, Top-down view of the CI domain from the mixed nucleotide state CI structure reported in Abe et al.40. with the nucleotides observed at each interface labeled. Schematic depictions of the specific atomic interactions with the 2′ hydroxyls as well as residues K224 and R226 observed at each interface exhibiting either two (b), one (c) or no (d) electrostatic interactions with the nucleotide phosphates.
Supplementary information
Supplementary Information
Supplementary Fig. 1–3 and Tables 1–6.
Supplementary Data 1
Raw data for binding and ATPase assays from supplementary figures.
Supplementary Software 1
DynaFit scripts for thermodynamic modeling.
Source data
Source Data Fig. 1
Titration curves and Kd,app values from Fig. 1.
Source Data Fig. 3
Kd,app values from Fig. 3.
Source Data Fig. 4
Kd,app values from Fig. 4.
Source Data Fig. 5
Cooperativity indices, Kd,app values, ATPase kcat values from Fig. 5.
Source Data Fig. 6
In vitro and in vivo oscillator time courses from Fig. 6.
Source Data Extended Data Fig. 5
Uncropped gel photo from Extended Data Fig. 5.
Source Data Extended Data Fig. 5
Densitometry values for KaiC phosphorylation from Extended Data Fig. 5.
Source Data Extended Data Fig. 9
Thermodynamic modeling outputs and cooperativity indices from Extended Data Fig. 9.
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Swan, J.A., Sandate, C.R., Chavan, A.G. et al. Coupling of distant ATPase domains in the circadian clock protein KaiC. Nat Struct Mol Biol 29, 759–766 (2022). https://doi.org/10.1038/s41594-022-00803-w
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DOI: https://doi.org/10.1038/s41594-022-00803-w
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