Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Coupling of distant ATPase domains in the circadian clock protein KaiC

Nature Structural & Molecular Biology volume 29, pages 759–766 (2022)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Daytime and nighttime phosphomimetics confer distinct biochemical activities and global conformations to KaiC.
Fig. 2: Overview of three-dimensional models obtained for daytime and nighttime KaC variants.
Fig. 3: Nucleotide interactions at CII-CII interfaces govern the transition between extended and compressed conformations.
Fig. 4: Compression of the cis CI-CII interface primes nighttime KaiC for KaiB association.
Fig. 5: Interactions in the CI nucleotide binding pocket link ATP hydrolysis to cooperative KaiB recruitment.
Fig. 6: Functional characterization of CI active site mutants.
Fig. 7: Model for regulation of KaiB association by KaiC phosphostate.

Similar content being viewed by others

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.

References

  1. Ishiura, M. et al. Expression of a gene cluster kaiABC as a circadian feedback process in cyanobacteria. Science 281, 1519–1523 (1998).

    Article  CAS  PubMed  Google Scholar 

  2. Diamond, S., Jun, D., Rubin, B. E. & Golden, S. S. The circadian oscillator in Synechococcus elongatus controls metabolite partitioning during diurnal growth. Proc. Natl Acad. Sci. USA 112, E1916–E1925 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Pattanayak, G. K., Phong, C. & Rust, M. J. Rhythms in energy storage control the ability of the cyanobacterial circadian clock to reset. Curr. Biol. 24, 1934–1938 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Hayashi, F. et al. Roles of two ATPase-motif-containing domains in cyanobacterial circadian clock protein KaiC. J. Biol. Chem. 279, 52331–52337 (2004).

    Article  CAS  PubMed  Google Scholar 

  5. Rust, M. J., Markson, J. S., Lane, W. S., Fisher, D. S. & O’Shea, E. K. Ordered phosphorylation governs oscillation of a three-protein circadian clock. Science 318, 809–812 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Nishiwaki, T. et al. A sequential program of dual phosphorylation of KaiC as a basis for circadian rhythm in cyanobacteria. EMBO J. 26, 4029–4037 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Kim, Y. I., Dong, G., Carruthers, C. W. Jr., Golden, S. S. & LiWang, A. The day/night switch in KaiC, a central oscillator component of the circadian clock of cyanobacteria. Proc. Natl Acad. Sci. USA 105, 12825–12830 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Chang, Y. G., Tseng, R., Kuo, N. W. & LiWang, A. Rhythmic ring-ring stacking drives the circadian oscillator clockwise. Proc. Natl Acad. Sci. USA 109, 16847–16851 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Snijder, J. et al. Insight into cyanobacterial circadian timing from structural details of the KaiB-KaiC interaction. Proc. Natl Acad. Sci. USA 111, 1379–1384 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Murakami, R. et al. Cooperative binding of KaiB to the KaiC Hexamer ensures accurate circadian clock oscillation in cyanobacteria. Int. J. Mol. Sci. https://doi.org/10.3390/ijms20184550 (2019).

  11. Chavan, A. G. et al. Reconstitution of an intact clock reveals mechanisms of circadian timekeeping. Science 374, eabd4453 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Chow, G. K. et al. A night-time edge site intermediate in the cyanobacterial circadian clock identified by EPR spectroscopy. J. Am. Chem. Soc. 144, 184–194 (2022).

    Article  CAS  PubMed  Google Scholar 

  13. Tseng, R. et al. Structural basis of the day-night transition in a bacterial circadian clock. Science 355, 1174–1180 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Chang, Y. G. et al. Circadian rhythms. A protein fold switch joins the circadian oscillator to clock output in cyanobacteria. Science 349, 324–328 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Chang, Y. G., Kuo, N. W., Tseng, R. & LiWang, A. Flexibility of the C-terminal, or CII, ring of KaiC governs the rhythm of the circadian clock of cyanobacteria. Proc. Natl Acad. Sci. USA 108, 14431–14436 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Tseng, R. et al. Cooperative KaiA-KaiB-KaiC interactions affect KaiB/SasA competition in the circadian clock of cyanobacteria. J. Mol. Biol. 426, 389–402 (2014).

    Article  CAS  PubMed  Google Scholar 

  17. Snijder, J. et al. Structures of the cyanobacterial circadian oscillator frozen in a fully assembled state. Science 355, 1181–1184 (2017).

    Article  CAS  PubMed  Google Scholar 

  18. Phong, C., Markson, J. S., Wilhoite, C. M. & Rust, M. J. Robust and tunable circadian rhythms from differentially sensitive catalytic domains. Proc. Natl Acad. Sci. USA 110, 1124–1129 (2013).

    Article  CAS  PubMed  Google Scholar 

  19. Terauchi, K. et al. ATPase activity of KaiC determines the basic timing for circadian clock of cyanobacteria. Proc. Natl Acad. Sci. USA 104, 16377–16381 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Ito-Miwa, K., Furuike, Y., Akiyama, S. & Kondo, T. Tuning the circadian period of cyanobacteria up to 6.6 days by the single amino acid substitutions in KaiC. Proc. Natl Acad. Sci. USA 117, 20926–20931 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Pattanayek, R., Xu, Y., Lamichhane, A., Johnson, C. H. & Egli, M. An arginine tetrad as mediator of input-dependent and input-independent ATPases in the clock protein KaiC. Acta Crystallogr. D. Biol. Crystallogr. 70, 1375–1390 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Dong, G. et al. Elevated ATPase activity of KaiC applies a circadian checkpoint on cell division in Synechococcus elongatus. Cell 140, 529–539 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Murakami, R. et al. The roles of the dimeric and tetrameric structures of the clock protein KaiB in the generation of circadian oscillations in cyanobacteria. J. Biol. Chem. 287, 29506–29515 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Murayama, Y. et al. Tracking and visualizing the circadian ticking of the cyanobacterial clock protein KaiC in solution. EMBO J. 30, 68–78 (2011).

    Article  CAS  PubMed  Google Scholar 

  25. Mukaiyama, A. et al. Conformational rearrangements of the C1 ring in KaiC measure the timing of assembly with KaiB. Sci. Rep. 8, 8803 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Oyama, K., Azai, C., Matsuyama, J. & Terauchi, K. Phosphorylation at Thr432 induces structural destabilization of the CII ring in the circadian oscillator KaiC. FEBS Lett. 592, 36–45 (2018).

    Article  CAS  PubMed  Google Scholar 

  27. Pattanayek, R. et al. Structures of KaiC circadian clock mutant proteins: a new phosphorylation site at T426 and mechanisms of kinase, ATPase and phosphatase. PLoS ONE 4, e7529 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Ito, H. et al. Autonomous synchronization of the circadian KaiC phosphorylation rhythm. Nat. Struct. Mol. Biol. 14, 1084–1088 (2007).

    Article  CAS  PubMed  Google Scholar 

  29. Hong, L., Vani, B. P., Thiede, E. H., Rust, M. J. & Dinner, A. R. Molecular dynamics simulations of nucleotide release from the circadian clock protein KaiC reveal atomic-resolution functional insights. Proc. Natl Acad. Sci. USA 115, E11475–E11484 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Oyama, K., Azai, C., Nakamura, K., Tanaka, S. & Terauchi, K. Conversion between two conformational states of KaiC is induced by ATP hydrolysis as a trigger for cyanobacterial circadian oscillation. Sci. Rep. 6, 32443 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Efremov, R. G., Leitner, A., Aebersold, R. & Raunser, S. Architecture and conformational switch mechanism of the ryanodine receptor. Nature 517, 39–43 (2015).

    Article  CAS  PubMed  Google Scholar 

  32. Pattanayek, R. et al. Visualizing a circadian clock protein: crystal structure of KaiC and functional insights. Mol. Cell 15, 375–388 (2004).

    Article  CAS  PubMed  Google Scholar 

  33. Nishiwaki, T. & Kondo, T. Circadian autodephosphorylation of cyanobacterial clock protein KaiC occurs via formation of ATP as intermediate. J. Biol. Chem. 287, 18030–18035 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Nishiwaki-Ohkawa, T., Kitayama, Y., Ochiai, E. & Kondo, T. Exchange of ADP with ATP in the CII ATPase domain promotes autophosphorylation of cyanobacterial clock protein KaiC. Proc. Natl Acad. Sci. USA 111, 4455–4460 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Leypunskiy, E. et al. The cyanobacterial circadian clock follows midday in vivo and in vitro. eLife https://doi.org/10.7554/eLife.23539 (2017).

  36. Jessop, M. et al. Structural insights into ATP hydrolysis by the MoxR ATPase RavA and the LdcI-RavA cage-like complex. Commun. Biol. 3, 46 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ye, Q. Z. et al. TRIP13 is a protein-remodeling AAA plus ATPase that catalyzes MAD2 conformation switching. eLife 4, e0736710.7554/eLife.07367 (2015).

  38. Glynn, S. E., Nager, A. R., Baker, T. A. & Sauer, R. T. Dynamic and static components power unfolding in topologically closed rings of a AAA+ proteolytic machine. Nat. Struct. Mol. Biol. 19, 616–622 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Nakajima, M., Ito, H. & Kondo, T. In vitro regulation of circadian phosphorylation rhythm of cyanobacterial clock protein KaiC by KaiA and KaiB. FEBS Lett. 584, 898–902 (2010).

    Article  CAS  PubMed  Google Scholar 

  40. Abe, J. et al. Circadian rhythms. Atomic-scale origins of slowness in the cyanobacterial circadian clock. Science 349, 312–316 (2015).

    Article  CAS  PubMed  Google Scholar 

  41. Erzberger, J. P. & Berger, J. M. Evolutionary relationships and structural mechanisms of AAA+ proteins. Annu. Rev. Biophys. Biomol. Struct. 35, 93–114 (2006).

    Article  CAS  PubMed  Google Scholar 

  42. Puri, N. et al. The molecular coupling between substrate recognition and ATP turnover in a AAA+ hexameric helicase loader. eLife https://doi.org/10.7554/eLife.64232 (2021).

  43. Gutu, A. & O’Shea, E. K. Two antagonistic clock-regulated histidine kinases time the activation of circadian gene expression. Mol. Cell 50, 288–294 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Kitayama, Y., Iwasaki, H., Nishiwaki, T. & Kondo, T. KaiB functions as an attenuator of KaiC phosphorylation in the cyanobacterial circadian clock system. EMBO J. 22, 2127–2134 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Cohen, S. E. et al. Dynamic localization of the cyanobacterial circadian clock proteins. Curr. Biol. 24, 1836–1844 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Ouyang, D. et al. Development and optimization of expression, purification, and ATPase assay of KaiC for medium-throughput screening of circadian clock mutants in cyanobacteria. Int. J. Mol. Sci. https://doi.org/10.3390/ijms20112789 (2019).

  47. Liu, H. & Naismith, J. H. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol. 8, 91 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Naydenova, K., Peet, M. J. & Russo, C. J. Multifunctional graphene supports for electron cryomicroscopy. Proc. Natl Acad. Sci. USA 116, 11718–11724 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Han, Y. et al. High-yield monolayer graphene grids for near-atomic resolution cryoelectron microscopy. Proc. Natl Acad. Sci. USA 117, 1009–1014 (2020).

    Article  CAS  PubMed  Google Scholar 

  50. Carragher, B. et al. Leginon: an automated system for acquisition of images from vitreous ice specimens. J. Struct. Biol. 132, 33–45 (2000).

    Article  CAS  PubMed  Google Scholar 

  51. Lander, G. C. et al. Appion: an integrated, database-driven pipeline to facilitate EM image processing. J. Struct. Biol. 166, 95–102 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Tan, Y. Z. et al. Addressing preferred specimen orientation in single-particle cryo-EM through tilting. Nat. Methods 14, 793–796 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Voss, N. R., Yoshioka, C. K., Radermacher, M., Potter, C. S. & Carragher, B. DoG Picker and TiltPicker: software tools to facilitate particle selection in single particle electron microscopy. J. Struct. Biol. 166, 205–213 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. https://doi.org/10.1016/j.jsb.2015.11.003 (2016).

  56. Roseman, A. M. FindEM–a fast, efficient program for automatic selection of particles from electron micrographs. J. Struct. Biol. 145, 91–99 (2004).

    Article  CAS  PubMed  Google Scholar 

  57. Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  58. Nicholls, R. A., Fischer, M., McNicholas, S. & Murshudov, G. N. Conformation-independent structural comparison of macromolecules with ProSMART. Acta Crystallogr. D. Biol. Crystallogr. 70, 2487–2499 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D. Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  CAS  Google Scholar 

  60. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D. Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Croll, T. I. ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallogr. D. Struct. Biol. 74, 519–530 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D. Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  PubMed  Google Scholar 

  63. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. UniProt, C. UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res. 49, D480–D489 (2021).

    Article  CAS  Google Scholar 

  65. Sievers, F. & Higgins, D. G. The clustal omega multiple alignment package. Methods Mol. Biol. 2231, 3–16 (2021).

    Article  CAS  PubMed  Google Scholar 

  66. Hayashi, F. et al. ATP-induced hexameric ring structure of the cyanobacterial circadian clock protein KaiC. Genes Cells 8, 287–296 (2003).

    Article  CAS  PubMed  Google Scholar 

  67. Bagshaw, C. R. Biomolecular Kinetics: A Step-By-Step Guide (CRC Press, Taylor & Francis Group, 2017).

  68. Heisler, J., Chavan, A., Chang, Y. G. & LiWang, A. Real-time in vitro fluorescence anisotropy of the cyanobacterial circadian clock. Methods Protoc. https://doi.org/10.3390/mps2020042 (2019).

  69. Ungerer, J. & Pakrasi, H. B. Cpf1 is a versatile tool for CRISPR genome editing across diverse species of cyanobacteria. Sci. Rep. 6, 39681 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Mackey, S. R. & Golden, S. S. Winding up the cyanobacterial circadian clock. Trends Microbiol. 15, 381–388 (2007).

    Article  CAS  PubMed  Google Scholar 

Download references

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.

Author information

Author notes

  1. These authors contributed equally: Jeffrey A. Swan, Colby R. Sandate.

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA, USA

    Jeffrey A. Swan, Alfred M. Freeberg, Diana Etwaru, Joseph G. Palacios & Carrie L. Partch

  2. Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA

    Colby R. Sandate & Gabriel C. Lander

  3. Department of Chemistry and Biochemistry, University of California, Merced, CA, USA

    Archana G. Chavan & Andy LiWang

  4. Center for Circadian Biology, University of California, San Diego, La Jolla, CA, USA

    Dustin C. Ernst, Susan S. Golden & Andy LiWang

  5. Division of Biological Sciences, University of California, San Diego, La Jolla, CA, USA

    Susan S. Golden & Carrie L. Partch

  6. Center for Cellular and Biomolecular Machines, University of California, Merced, CA, USA

    Andy LiWang

Authors
  1. Jeffrey A. Swan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Colby R. Sandate
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Archana G. Chavan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alfred M. Freeberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Diana Etwaru
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Dustin C. Ernst
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Joseph G. Palacios
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Susan S. Golden
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Andy LiWang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Gabriel C. Lander
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Carrie L. Partch
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding authors

Correspondence to Gabriel C. Lander or Carrie L. Partch.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editors: Beth Moorefield and Carolina Perdigoto, in collaboration with the Nature Structural & Molecular Biology team.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Source data

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.

Source data

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.

Reporting Summary

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.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41594-022-00803-w

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing