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:

Crystal structure of the C-terminal clock-oscillator domain of the cyanobacterial KaiA protein

Nature Structural & Molecular Biology volume 11, pages 623–631 (2004)Cite this article

Abstract

KaiA, KaiB and KaiC constitute the circadian clock machinery in cyanobacteria, and KaiA activates kaiBC expression whereas KaiC represses it. Here we show that KaiA is composed of three functional domains, the N-terminal amplitude-amplifier domain, the central period-adjuster domain and the C-terminal clock-oscillator domain. The C-terminal domain is responsible for dimer formation, binding to KaiC, enhancing KaiC phosphorylation and generating the circadian oscillations. The X-ray crystal structure at a resolution of 1.8 Å of the C-terminal clock-oscillator domain of KaiA from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 shows that residue His270, located at the center of a KaiA dimer concavity, is essential to KaiA function. KaiA binding to KaiC probably occurs via the concave surface. On the basis of the structure, we predict the structural roles of the residues that affect circadian oscillations.

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

Figure 1: Alignment of the deduced amino acid sequences of the C-terminal domain of KaiAs (residues 174–283 of T. elongatus KaiA) from 11 strains of cyanobacteria.
Figure 2: In vivo rhythm functions of the three KaiA domains.
Figure 3: Synechococcus KaiA dimerization assayed by gel filtration chromatography.
Figure 4: Binding activity of KaiA to KaiC.
Figure 5: Enhancement of KaiC phosphorylation by KaiA.
Figure 6: Overview of the structure of T. elongatus KaiA(174–283) (the C-terminal clock-oscillator domain).
Figure 7: The biophysical properties and in vitro biochemical functions of wild-type (WT) T. elongatus KaiA(174–283) (the C-terminal clock-oscillator domain) and T. elongatus KaiA(174–283) H270A, and the in vivo rhythm phenotypes of Synechococcus KaiA WT and Synechococcus KaiA H271A.
Figure 8: Bioluminescence rhythms of Synechococcus KaiA mutants.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

Protein Data Bank

References

  1. Dunlap, J.C. Molecular bases for circadian clocks. Cell 96, 271–290 (1999).

    Article  CAS  Google Scholar 

  2. Young, M.W. & Kay, S.A. Time zones: a comparative genetics of circadian clocks. Nat. Rev. Genet. 2, 702–715 (2001).

    Article  CAS  Google Scholar 

  3. Grobbelaar, N., Huang, T.C., Lin, H.Y. & Chow, T.J. Dinitrogen-fixing endogenous rhythm in Synechococcus RF-1. FEMS Microbiol. Lett. 37, 173–177 (1986).

    Article  CAS  Google Scholar 

  4. Mitsui, A. et al. Strategy by which nitrogen-fixing unicellular cyanobacteria grow photoautotrophically. Nature 323, 720–722 (1986).

    Article  CAS  Google Scholar 

  5. Kondo, T. et al. Circadian rhythms in prokaryotes: Luciferase as a reporter of circadian gene expression in cyanobacteria. Proc. Natl. Acad. Sci. USA 90, 5672–5676 (1993).

    Article  CAS  Google Scholar 

  6. Aoki, S., Kondo, T. & Ishiura, M. Circadian expression of the dnaK gene in the cyanobacterium Synechocystis sp. strain PCC 6803. J. Bacteriol. 177, 5606–5611 (1995).

    Article  CAS  Google Scholar 

  7. Chen, Y.B., Dominic, B., Mellon, M.T. & Zehr, J.P. Circadian rhythm of nitrogenase gene expression in the diazotrophic filamentous nonheterocystous cyanobacterium Trichodesmium sp. strain IMS 101. J. Bacteriol. 180, 3598–3605 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. Iwasaki, H., Taniguchi, Y., Ishiura, M. & Kondo, T. Physical interactions among circadian clock proteins KaiA, KaiB and KaiC in cyanobacteria. EMBO J. 18, 1137–1145 (1999).

    Article  CAS  Google Scholar 

  10. Hayashi, F. et al. Stoichiometric interactions between cyanobacterial clock proteins KaiA and KaiC. Biochem. Biophys. Res. Comm. 316, 195–202 (2004).

    Article  CAS  Google Scholar 

  11. Nishiwaki, T., Iwasaki, H., Ishiura, M. & Kondo, T. Nucleotide binding and autophosphorylation of the clock protein KaiC as a circadian timing process of cyanobacteria. Proc. Natl. Acad. Sci. USA 97, 495–499 (2000).

    Article  CAS  Google Scholar 

  12. Taniguchi, Y. et al. Two KaiA-binding domains of cyanobacterial circadian clock protein KaiC. FEBS Lett. 496, 86–90 (2001).

    Article  CAS  Google Scholar 

  13. Nishimura, H. et al. Mutations in KaiA, a clock protein, extend the period of circadian rhythm in the cyanobacterium Synechococcus elongatus PCC 7942. Microbiology 148, 2903–2909 (2002).

    Article  CAS  Google Scholar 

  14. Iwasaki, H., Nishiwaki, T., Kitayama, Y., Nakajima, M. & Kondo, T. KaiA-stimulated KaiC phosphorylation in circadian timing loops in cyanobacteria. Proc. Natl. Acad. Sci. USA 99, 15788–15793 (2002).

    Article  CAS  Google Scholar 

  15. Yamaoka, T., Satoh, K. & Katoh, S. Photosynthetic activities of a thermophilic blue-green alga. Plant Cell Physiol. 19, 943–954 (1978).

    Article  CAS  Google Scholar 

  16. Van Baalen, C. Studies on marine blue-green algae. Bot. Mar. 4, 129–139 (1962).

    Article  Google Scholar 

  17. Rippka, R., Deruelles, J., Waterbury, J.B., Herdman, M. & Stanier, R.Y. Generic assignments, strain histories, and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 111, 1–61 (1979).

    Google Scholar 

  18. Marquardt, J., Senger, H., Miyashita, H., Miyachi, S. & Morschel, E. Isolation and characterization of biliprotein aggregates from Acaryochloris marina, a Prochloron-like prokaryote containing mainly chlorophyll d. FEBS Lett. 410, 428–432 (1997).

    Article  CAS  Google Scholar 

  19. Hirose, M. Energy supply system for the gliding movement of hormogonia of the cyanobacterium Nostoc cycadae. Plant Cell Physiol. 28, 584–597 (1987).

    Google Scholar 

  20. Kaneko, T. et al. Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120. DNA Res. 8, 205–213 (2001).

    Article  CAS  Google Scholar 

  21. Hendrickson, W.A., Horton, J.R. & LeMaster, D.M. Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure. EMBO J. 9, 1665–1672 (1990).

    Article  CAS  Google Scholar 

  22. Vakonakis, I. et al. NMR structure of the KaiC-interacting C-terminal domain of KaiA, a circadian clock protein: Implications for KaiA-KaiC interaction. Proc. Natl. Acad. Sci. USA 101, 1479–1484 (2004).

    Article  CAS  Google Scholar 

  23. Ye, S., Vakonakis, I., Ioerger, T.R., LiWang, A.C. & Sacchettini, J.C. Crystal structure of circadian clock protein KaiA from Synechococcus elongatus. J. Biol. Chem. 279, 20511–20518 (2004).

    Article  CAS  Google Scholar 

  24. Onai, K., Morishita, M., Itoh, S., Okamoto, K., & Ishiura, M. Circadian rhythms in the thermophilic cyanobacterium Thermosynechococcus elongatus: Compensation of period length over wide temperature range. J. Bacteriol. (in the press).

  25. Williams, S.B., Vakonakis, I., Golden, S.S. & LiWang, A.C. Structure and function from the circadian clock protein KaiA of Synechococcus elongatus: A potential clock input mechanism. Proc. Natl. Acad. Sci. USA 99, 15357–15362 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  27. Onai, K., Morishita, M., Kaneko, T., Tabata, S. & Ishiura, M. Natural transformation of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1: A simple and efficient method for gene transfer. Mol. Genet. Genom. 271, 50–59 (2004).

    Article  CAS  Google Scholar 

  28. Pflugrath, J.W. The finer things in X-ray diffraction data collection. Acta Crystallogr. D 55, 1718–1725 (1999).

    Article  CAS  Google Scholar 

  29. Terwilliger, T.C. & Berendzen, J. Automated structure solution for MIR and MAD. Acta Crystallogr. D 55, 849–861 (1999).

    Article  CAS  Google Scholar 

  30. Perrakis, A., Morris, R. & Lamzin, V.S. Automated protein model building combined with iterative structure refinement. Nature Struct. Biol. 6, 458–463 (1999).

    Article  CAS  Google Scholar 

  31. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997).

    Article  CAS  Google Scholar 

  32. Roussel, A. & Cambillau, C. Turbo-Frodo Manual (AFMB-CNRS, Marseille, France, 1996).

    Google Scholar 

  33. Thompson, J.D., Higgins, D.G. & Gibson, T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680 (1994).

    Article  CAS  Google Scholar 

  34. Kraulis, P.J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991).

    Article  Google Scholar 

  35. Merritt, E.A. & Bacon, D.J. Raster3D: photorealistic molecular graphics. Methods Enzymol. 277, 505–524 (1997).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J.-R. Shen and Y. Inoue (RIKEN Harima Institute) for T. vulcanus, M. Ikeuchi for T. elongatus, S. Itoh (Nagoya University) for Synechococcus 7002 and A. marina, M. Hirose (Wakayama University) for N. cycadae, and C.P. Wolk (Michigan State University) for Anabaena. We thank K. Onai and M. Morishita for their help in rhythm analysis. We thank M. Yamamoto (RIKEN Harima Institute) for X-ray diffraction data collection on the RIKEN beamline BL26B1, B2 at SPring-8. We thank M. Bloom (SciWrite Biomedical Writing & Editing Services) for professional editing. This research was supported by grants from the Japanese Ministry of Education, Science and Culture (MEXT), the Naito Foundation, PROBRAIN, the Future of Japan Society for the Promotion of Science, the Japan Space Forum, the Aichi Science and Technology Foundation, and the National Project on Protein Structural and Functional Analyses by MEXT to M.I. The Division of Biological Science, Graduate School of Science, Nagoya University, was supported by a 21st Century Center of Excellence grant from MEXT.

Author information

Author notes

  1. Tatsuya Uzumaki, Masayasu Fujita and Toru Nakatsu: These authors contributed equally to this work.

Authors and Affiliations

  1. Center for Gene Research, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, 464-8602, Nagoya, Japan

    Tatsuya Uzumaki, Masayasu Fujita, Fumio Hayashi, Noriyo Itoh & Masahiro Ishiura

  2. BRAIN, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, 464-8602, Nagoya, Japan

    Tatsuya Uzumaki, Fumio Hayashi & Masahiro Ishiura

  3. Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, 464-8602, Nagoya, Japan

    Masayasu Fujita, Noriyo Itoh & Masahiro Ishiura

  4. Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, 606-8501, Kyoto, Japan

    Toru Nakatsu & Hiroaki Kato

  5. RIKEN Harima Institute, 1-1-1 Kohto, Mikazuki-cho, Sayo, 679-5148, Hyogo, Japan

    Toru Nakatsu, Hiroyuki Shibata & Hiroaki Kato

Authors
  1. Tatsuya Uzumaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Masayasu Fujita
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Toru Nakatsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Fumio Hayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hiroyuki Shibata
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Noriyo Itoh
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hiroaki Kato
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Masahiro Ishiura
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Hiroaki Kato or Masahiro Ishiura.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Alignment of the deduced amino acid sequences of KaiAs from 11 strains of cyanobacteria. (PDF 35 kb)

Supplementary Fig. 2

Rescue of circadian bioluminescence rhythms in a Synechococcus kaiA-null mutant strain by transferring the T. elongatus kaiA gene into the strain. (PDF 30 kb)

Supplementary Fig. 3

Density map (stereo view) showing the atomic arrangement in the region of the functionally essential residue His270. (PDF 53 kb)

Supplementary Fig. 4

The biophysical properties of T. elongatus KaiA(174–283) WT (the C-terminal clock-oscillator domain) and T. elongatus KaiA(174–283) H270A. (PDF 31 kb)

Supplementary Fig. 5

Differences in the in vivo rhythm assay procedures used between a previous report13 and this study. (PDF 34 kb)

Supplementary Fig. 6

Superposition of the crystal structure of KaiA(174–283) with ThKaiA180C NMR solution structure22. (PDF 57 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Uzumaki, T., Fujita, M., Nakatsu, T. et al. Crystal structure of the C-terminal clock-oscillator domain of the cyanobacterial KaiA protein. Nat Struct Mol Biol 11, 623–631 (2004). https://doi.org/10.1038/nsmb781

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb781

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