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Structure of green-type Rubisco activase from tobacco

Nature Structural & Molecular Biology volume 18, pages 1366–1370 (2011)Cite this article

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Abstract

Rubisco, the enzyme that catalyzes the fixation of atmospheric CO2 in photosynthesis, is subject to inactivation by inhibitory sugar phosphates. Here we report the 2.95-Å crystal structure of Nicotiana tabacum Rubisco activase (Rca), the enzyme that facilitates the removal of these inhibitors. Rca from tobacco has a classical AAA+-protein domain architecture. Although Rca populates a range of oligomeric states when in solution, it forms a helical arrangement with six subunits per turn when in the crystal. However, negative-stain electron microscopy of the active mutant R294V suggests that Rca functions as a hexamer. The residues determining species specificity for Rubisco are located in a helical insertion of the C-terminal domain and probably function in conjunction with the N-domain in Rubisco recognition. Loop segments exposed toward the central pore of the hexamer are required for the ATP-dependent remodeling of Rubisco, resulting in the release of inhibitory sugar.

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Figure 1: Structural and functional analysis of Rubisco activase.
Figure 2: Oligomeric association of Rubisco activase subunits.
Figure 3: Structural and functional analysis of the Rca oligomeric state.
Figure 4: Structure and function of the Rca hexamer pore.

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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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Salvucci, M.E. & Crafts-Brandner, S.J. Relationship between the heat tolerance of photosynthesis and the thermal stability of rubisco activase in plants from contrasting thermal environments. Plant Physiol. 134, 1460–1470 (2004).

    Article  CAS  Google Scholar 

  6. Kurek, I. et al. Enhanced thermostability of Arabidopsis Rubisco activase improves photosynthesis and growth rates under moderate heat stress. Plant Cell 19, 3230–3241 (2007).

    Article  CAS  Google Scholar 

  7. Wang, Z.Y., Snyder, G.W., Esau, B.D., Portis, A.R. Jr. & Ogren, W.L. Species-dependent variation in the interaction of substrate-bound ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and rubisco activase. Plant Physiol. 100, 1858–1862 (1992).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. Li, C., Salvucci, M.E. & Portis, A.R. Two residues of Rubisco activase involved in recognition of the Rubisco substrate. J. Biol. Chem. 280, 24864–24869 (2005).

    Article  CAS  Google Scholar 

  10. van de Loo, F.J. & Salvucci, M.E. Activation of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) involves Rubisco activase Trp16. Biochemistry 35, 8143–8148 (1996).

    Article  CAS  Google Scholar 

  11. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  13. Guo, F., Maurizi, M.R., Esser, L. & Xia, D. Crystal structure of ClpA, an Hsp100 chaperone and regulator of ClpAP protease. J. Biol. Chem. 277, 46743–46752 (2002).

    Article  CAS  Google Scholar 

  14. Kim, D.Y. & Kim, K.K. Crystal structure of ClpX molecular chaperone from Helicobacter pylori. J. Biol. Chem. 278, 50664–50670 (2003).

    Article  CAS  Google Scholar 

  15. Blayney, M.J., Whitney, S.M. & Beck, J.L. NanoESI mass spectrometry of Rubisco and Rubisco activase structures and their interactions with nucleotides and sugar phosphates. J. Am. Soc. Mass Spectrom. 22, 1588–1601 (2011).

    Article  CAS  Google Scholar 

  16. Li, C., Wang, D. & Portis, A.R. Jr. Identification of critical arginine residues in the functioning of Rubisco activase. Arch. Biochem. Biophys. 450, 176–182 (2006).

    Article  CAS  Google Scholar 

  17. Barta, C., Dunkle, A.M., Wachter, R.M. & Salvucci, M.E. Structural changes associated with the acute thermal instability of Rubisco activase. Arch. Biochem. Biophys. 499, 17–25 (2010).

    Article  CAS  Google Scholar 

  18. 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).

    Article  CAS  Google Scholar 

  19. 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).

    Article  CAS  Google Scholar 

  20. 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  Google Scholar 

  21. Mueller-Cajar, O. et al. Structure and function of the AAA+ protein CbbX, a red-type Rubisco activase. Nature doi:10.1038/nature10568 (2011).

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

    Article  CAS  Google Scholar 

  23. Servaites, J.C. Crystalline ribulose bisphosphate carboxylase/oxygenase of high integrity and catalytic activity from Nicotiana tabacum. Arch. Biochem. Biophys. 238, 154–160 (1985).

    Article  CAS  Google Scholar 

  24. Goloubinoff, P., Gatenby, A.A. & Lorimer, G.H. GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli. Nature 337, 44–47 (1989).

    Article  CAS  Google Scholar 

  25. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  27. Barta, C., Carmo-Silva, A.E. & Salvucci, M.E. Rubisco activase activity assays. Methods Mol. Biol. 684, 375–382 (2011).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  29. 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 

  30. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  32. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  35. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr 50, 760–763 (1994).

  36. Evans, P.R. Scala. in Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography Vol. 33, 22–24 (Daresbury Laboratory, Warrington, UK, 1997).

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

    Article  Google Scholar 

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

    Article  Google Scholar 

  39. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  42. 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).

    Article  CAS  Google Scholar 

  43. 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. Crystallogr. 26, 283–291 (1993).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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Acknowledgements

We thank T. Wauer and L. Popilka for assistance in protein purification and enzyme assays, the Max Planck Institute of Biochemistry Crystallization Facility for support during screening, as well as the Joint Structural Biology Group staff at the European Synchrotron Radiation Facility. Tobacco leaves for the purification of Rubisco were a gift of H.-U. Koob and S. Kirchner (Ludwig-Maximilians-Universität München). The pHUENtRca plasmid was a gift from S. Whitney (Australian National University). We thank the Deutsche Forschungsgemeinschaft (DFG) (SFB 594; DFG grant WE4628/1 to P.W.) and the Körber Foundation for financial support.

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Authors and Affiliations

  1. Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany

    Mathias Stotz, Oliver Mueller-Cajar, F Ulrich Hartl, Andreas Bracher & Manajit Hayer-Hartl

  2. Department of Biochemistry, Gene Center Munich, Ludwig-Maximilians-Universität München, Munich, Germany

    Susanne Ciniawsky & Petra Wendler

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  1. Mathias Stotz
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Contributions

M.S. obtained the Rca crystals and solved the structure together with A.B. M.S. carried out all the biochemical experiments, with help from O.M.-C. The EM and 3D-image analysis were done by S.C. and P.W. All authors contributed to data interpretation and manuscript preparation. M.S., A.B., F.U.H. and M.H.-H. wrote the paper.

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Correspondence to Andreas Bracher or Manajit Hayer-Hartl.

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The authors declare no competing financial interests.

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Stotz, M., Mueller-Cajar, O., Ciniawsky, S. et al. Structure of green-type Rubisco activase from tobacco. Nat Struct Mol Biol 18, 1366–1370 (2011). https://doi.org/10.1038/nsmb.2171

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