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.

Structure and function of the AAA+ protein CbbX, a red-type Rubisco activase

Abstract

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the fixation of atmospheric CO2 in photosynthesis, but tends to form inactive complexes with its substrate ribulose 1,5-bisphosphate (RuBP). In plants, Rubisco is reactivated by the AAA+ (ATPases associated with various cellular activities) protein Rubisco activase (Rca), but no such protein is known for the Rubisco of red algae. Here we identify the protein CbbX as an activase of red-type Rubisco. The 3.0-Å crystal structure of unassembled CbbX from Rhodobacter sphaeroides revealed an AAA+ protein architecture. Electron microscopy and biochemical analysis showed that ATP and RuBP must bind to convert CbbX into functionally active, hexameric rings. The CbbX ATPase is strongly stimulated by RuBP and Rubisco. Mutational analysis suggests that CbbX functions by transiently pulling the carboxy-terminal peptide of the Rubisco large subunit into the hexamer pore, resulting in the release of the inhibitory RuBP. Understanding Rubisco activation may facilitate efforts to improve CO2 uptake and biomass production by photosynthetic organisms.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: CbbX functions as a Rubisco activase.
Figure 2: Negative-stain electron microscopy of CbbX.
Figure 3: Crystal structure and hexamer model of CbbX.
Figure 4: Structural and functional analysis of CbbX mechanism.
Figure 5: Model of Rubisco activation by CbbX.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factor amplitudes for CbbX crystal structures are deposited in the Protein Data Bank (PDB) under accession codes 3SYL and 3SYK; the hexamer model and the electron microscopy density are deposited in the PDB under accession code 3ZUH and in the Electron Microscopy Database (http://www.ebi.ac.uk/pdbe/emdb/) under accession code EMD-1932, respectively.

References

  1. Spreitzer, R. J. & Salvucci, M. E. Rubisco: structure, regulatory interactions, and possibilities for a better enzyme. Annu. Rev. Plant Biol. 53, 449–475 (2002)

    CAS  Article  Google Scholar 

  2. Andersson, I. & Backlund, A. Structure and function of Rubisco. Plant Physiol. Biochem. 46, 275–291 (2008)

    CAS  Article  Google Scholar 

  3. Tabita, F. R. Microbial ribulose 1,5-bisphosphate carboxylase/oxygenase: a different perspective. Photosynth. Res. 60, 1–28 (1999)

    CAS  Article  Google Scholar 

  4. Tabita, F. R., Satagopan, S., Hanson, T. E., Kreel, N. E. & Scott, S. S. Distinct form I, II, III, and IV Rubisco proteins from the three kingdoms of life provide clues about Rubisco evolution and structure/function relationships. J. Exp. Bot. 59, 1515–1524 (2008)

    CAS  Article  Google Scholar 

  5. Badger, M. R. & Bek, E. J. Multiple Rubisco forms in proteobacteria: their functional significance in relation to CO2 acquisition by the CBB cycle. J. Exp. Bot. 59, 1525–1541 (2008)

    CAS  Article  Google Scholar 

  6. Whitney, S. M., Baldet, P., Hudson, G. S. & Andrews, T. J. Form I Rubiscos from non-green algae are expressed abundantly but not assembled in tobacco chloroplasts. Plant J. 26, 535–547 (2001)

    CAS  Article  Google Scholar 

  7. Falkowski, P. G. et al. The evolution of modern eukaryotic phytoplankton. Science 305, 354–360 (2004)

    CAS  Article  ADS  Google Scholar 

  8. Lorimer, G. H., Badger, M. R. & Andrews, T. J. The activation of ribulose-1,5-bisphosphate carboxylase by carbon dioxide and magnesium ions. Equilibria, kinetics, a suggested mechanism, and physiological implications. Biochemistry 15, 529–536 (1976)

    CAS  Article  Google Scholar 

  9. Jordan, D. B. & Chollet, R. Inhibition of ribulose bisphosphate carboxylase by substrate ribulose 1,5-bisphosphate. J. Biol. Chem. 258, 13752–13758 (1983)

    CAS  PubMed  Google Scholar 

  10. Portis, A. R., Jr Rubisco activase—Rubisco’s catalytic chaperone. Photosynth. Res. 75, 11–27 (2003)

    CAS  Article  Google Scholar 

  11. Hanson, P. I. & Whiteheart, S. W. AAA+ proteins: have engine, will work. Nature Rev. Mol. Cell Biol. 6, 519–529 (2005)

    CAS  Article  Google Scholar 

  12. Pearce, F. G. Catalytic by-product formation and ligand binding by ribulose bisphosphate carboxylases from different phylogenies. Biochem. J. 399, 525–534 (2006)

    CAS  Article  Google Scholar 

  13. Gibson, J. L. & Tabita, F. R. Analysis of the cbbXYZ operon in Rhodobacter sphaeroides. J. Bacteriol. 179, 663–669 (1997)

    CAS  Article  Google Scholar 

  14. Maier, U. G., Fraunholz, M., Zauner, S., Penny, S. & Douglas, S. A nucleomorph-encoded CbbX and the phylogeny of RuBisCo regulators. Mol. Biol. Evol. 17, 576–583 (2000)

    CAS  Article  Google Scholar 

  15. Fujita, K., Tanaka, K., Sadaie, Y. & Ohta, N. Functional analysis of the plastid and nuclear encoded CbbX proteins of Cyanidioschyzon merolae. Genes Genet. Syst. 83, 135–142 (2008)

    CAS  Article  Google Scholar 

  16. Bowien, B. & Kusian, B. Genetics and control of CO2 assimilation in the chemoautotroph Ralstonia eutropha. Arch. Microbiol. 178, 85–93 (2002)

    CAS  Article  Google Scholar 

  17. Saschenbrecker, S. et al. Structure and function of RbcX, an assembly chaperone for hexadecameric Rubisco. Cell 129, 1189–1200 (2007)

    CAS  Article  Google Scholar 

  18. Liu, C. et al. Coupled chaperone action in folding and assembly of hexadecameric Rubisco. Nature 463, 197–202 (2010)

    CAS  Article  ADS  Google Scholar 

  19. Gibson, J. L. & Tabita, F. R. Activation of ribulose 1,5-bisphosphate carboxylase from Rhodopseudomonas sphaeroides: probable role of the small subunit. J. Bacteriol. 140, 1023–1027 (1979)

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Robinson, S. P. & Portis, A. R., Jr Adenosine triphosphate hydrolysis by purified rubisco activase. Arch. Biochem. Biophys. 268, 93–99 (1989)

    CAS  Article  Google Scholar 

  21. Sugawara, H. et al. Crystal structure of carboxylase reaction-oriented ribulose 1,5-bisphosphate carboxylase/oxygenase from a thermophilic red alga, Galdieria partita. J. Biol. Chem. 274, 15655–15661 (1999)

    CAS  Article  Google Scholar 

  22. Okano, Y. et al. X-ray structure of Galdieria Rubisco complexed with one sulfate ion per active site. FEBS Lett. 527, 33–36 (2002)

    CAS  Article  ADS  Google Scholar 

  23. Von Caemmerer, S. & Edmondson, D. L. Relationship between steady-state gas exchange in vivo ribulose bisphosphate carboxylase activity and some carbon reduction cycle intermediates in Raphanus sativus. Aust. J. Plant Physiol. 13, 669–688 (1986)

    CAS  Google Scholar 

  24. Sousa, M. C. et al. Crystal and solution structures of an HslUV protease–chaperone complex. Cell 103, 633–643 (2000)

    CAS  Article  Google Scholar 

  25. Massey, T. H., Mercogliano, C. P., Yates, J., Sherratt, D. J. & Lowe, J. Double-stranded DNA translocation: structure and mechanism of hexameric FtsK. Mol. Cell 23, 457–469 (2006)

    CAS  Article  Google Scholar 

  26. Matias, P. M., Gorynia, S., Donner, P. & Carrondo, M. A. Crystal structure of the human AAA+ protein RuvBL1. J. Biol. Chem. 281, 38918–38929 (2006)

    CAS  Article  Google Scholar 

  27. Davies, J. M., Brunger, A. T. & Weis, W. I. Improved structures of full-length p97, an AAA ATPase: implications for mechanisms of nucleotide-dependent conformational change. Structure 16, 715–726 (2008)

    CAS  Article  Google Scholar 

  28. Glynn, S. E., Martin, A., Nager, A. R., Baker, T. A. & Sauer, R. T. Structures of asymmetric ClpX hexamers reveal nucleotide-dependent motions in a AAA+ protein-unfolding machine. Cell 139, 744–756 (2009)

    CAS  Article  Google Scholar 

  29. Weibezahn, J. et al. Thermotolerance requires refolding of aggregated proteins by substrate translocation through the central pore of ClpB. Cell 119, 653–665 (2004)

    CAS  Article  Google Scholar 

  30. Hinnerwisch, J., Fenton, W. A., Furtak, K. J., Farr, G. W. & Horwich, A. L. Loops in the central channel of ClpA chaperone mediate protein binding, unfolding, and translocation. Cell 121, 1029–1041 (2005)

    CAS  Article  Google Scholar 

  31. Martin, A., Baker, T. A. & Sauer, R. T. Pore loops of the AAA+ ClpX machine grip substrates to drive translocation and unfolding. Nature Struct. Mol. Biol. 15, 1147–1151 (2008)

    CAS  Article  Google Scholar 

  32. Roll-Mecak, A. & Vale, R. D. Structural basis of microtubule severing by the hereditary spastic paraplegia protein spastin. Nature 451, 363–367 (2008)

    CAS  Article  ADS  Google Scholar 

  33. Andersson, I. Catalysis and regulation in Rubisco. J. Exp. Bot. 59, 1555–1568 (2008)

    CAS  Article  Google Scholar 

  34. Catanzariti, A.-M., Soboleva, T. A., Jans, D. A., Board, P. G. & Baker, R. T. An efficient system for high-level expression and easy purification of authentic recombinant proteins. Protein Sci. 13, 1331–1339 (2004)

    CAS  Article  Google Scholar 

  35. Baker, R. T. et al. Using deubiquitylating enzymes as research tools. Methods Enzymol. 398, 540–554 (2005)

    CAS  Article  Google Scholar 

  36. Esau, B. D., Snyder, G. W. & Portis, A. R., Jr Differential effects of N- and C-terminal deletions on the two activities of rubisco activase. Arch. Biochem. Biophys. 326, 100–105 (1996)

    CAS  Article  Google Scholar 

  37. Kreuzer, K. N. & Jongeneel, C. V. Escherichia coli phage T4 topoisomerase. Methods Enzymol. 100, 144–160 (1983)

    CAS  Article  Google Scholar 

  38. Parry, M. A. J., Keys, A. J., Madgwick, P. J., Carmo-Silva, A. E. & Andralojc, P. J. Rubisco regulation: a role for inhibitors. J. Exp. Bot. 59, 1569–1580 (2008)

    CAS  Article  Google Scholar 

  39. Pitcher, D. G., Saunders, N. A. & Owen, R. J. Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett. Appl. Microbiol. 8, 151–156 (1989)

    CAS  Article  Google Scholar 

  40. Gibson, J. L., Falcone, D. L. & Tabita, F. R. Nucleotide sequence, transcriptional analysis, and expression of genes encoded within the form I CO2 fixation operon of Rhodobacter sphaeroides. J. Biol. Chem. 266, 14646–14653 (1991)

    CAS  PubMed  Google Scholar 

  41. Guzman, L. M., Belin, D., Carson, M. J. & Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose p-BAD promoter. J. Bacteriol. 177, 4121–4130 (1995)

    CAS  Article  Google Scholar 

  42. Edmondson, D. L., Badger, M. R. & Andrews, T. J. A kinetic characterization of slow inactivation of ribulosebisphosphate carboxylase during catalysis. Plant Physiol. 93, 1376–1382 (1990)

    CAS  Article  Google Scholar 

  43. Smith, J. M. Ximdisp—a visualization tool to aid structure determination from electron microscope images. J. Struct. Biol. 125, 223–228 (1999)

    CAS  Article  Google Scholar 

  44. Mindell, J. A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003)

    Article  Google Scholar 

  45. Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199 (1996)

    CAS  Article  Google Scholar 

  46. Shaikh, T. R. et al. SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs. Nature Protocols 3, 1941–1974 (2008)

    CAS  Article  Google Scholar 

  47. van Heel, M., Harauz, G., Orlova, E. V., Schmidt, R. & Schatz, M. A new generation of the IMAGIC image processing system. J. Struct. Biol. 116, 17–24 (1996)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  49. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010)

    CAS  Article  Google Scholar 

  50. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62, 72–82 (2006)

    Article  Google Scholar 

  51. Evans, P. R. Scala. CCP4 ESF-EACBM Newsl. Prot. Crystallogr. 33, 22–24 (1997)

    Google Scholar 

  52. Collaborative Computational Project No. 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994)

  53. French, G. & Wilson, K. On the treatment of negative intensity observations. Acta Crystallogr. A 34, 517–525. (1978)

  54. Van Duyne, G. D., Standaert, R. F., Karplus, P. A., Schreiber, S. L. & Clardy, J. Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin. J. Mol. Biol. 229, 105–124 (1993)

    CAS  Article  Google Scholar 

  55. Schneider, T. R. & Sheldrick, G. M. Substructure solution with SHELXD. Acta Crystallogr. D Biol. Crystallogr. 58, 1772–1779 (2002)

    Article  Google Scholar 

  56. de la Fortelle, E. & Bricogne, G. Maximum-likelihood heavy atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 472–494 (1997)

    CAS  Article  Google Scholar 

  57. Terwilliger, T. C. Maximum-likelihood density modification. Acta Crystallogr. D Biol. Crystallogr. 56, 965–972 (2000)

    CAS  Article  Google Scholar 

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

    Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  60. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283–291 (1993)

    CAS  Article  Google Scholar 

  61. Kleywegt, G. T. & Jones, T. A. A super position. CCP4/ESF-EACBM Newsl. Prot. Crystallogr. 31, 9–14 (1994)

    Google Scholar 

  62. Gouet, P., Courcelle, E., Stuart, D. I. & Metoz, F. ESPript: multiple sequence alignments in PostScript. Bioinformatics 15, 305–308 (1999)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank S. Kaplan for providing the R. sphaeroides strain 2.4.1, S. Whitney for providing the pHue protein expression system, and R. Lange and N. Wischnewski for technical assistance. Support by the Max Planck Institute of Biochemistry (MPIB) Core Facility, the MPIB Crystallization Facility and the Joint Structural Biology Group staff at the European Synchrotron Radiation Facility beamlines is gratefully acknowledged. We thank the Deutsche Forschungsgemeinschaft (DFG) (SFB 594; DFG grant WE4628/1 to P.W.) and the Körber Foundation for financial support.

Author information

Authors and Affiliations

Authors

Contributions

O.M.-C. designed and performed all the biochemical experiments. O.M.-C., M.S. and A.B. obtained the CbbX crystals and solved the structure. P.W. performed the electron microscopy and three-dimensional image analysis. All authors contributed to data interpretation and manuscript preparation. O.M.-C., A.B., F.U.H. and M.H.-H. wrote the manuscript.

Corresponding authors

Correspondence to Andreas Bracher or Manajit Hayer-Hartl.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Tables 1-2 and Supplementary Figures 1-5 with legends. (PDF 1551 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Mueller-Cajar, O., Stotz, M., Wendler, P. et al. Structure and function of the AAA+ protein CbbX, a red-type Rubisco activase. Nature 479, 194–199 (2011). https://doi.org/10.1038/nature10568

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature10568

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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