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Coupled chaperone action in folding and assembly of hexadecameric Rubisco


Form I Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase), a complex of eight large (RbcL) and eight small (RbcS) subunits, catalyses the fixation of atmospheric CO2 in photosynthesis. The limited catalytic efficiency of Rubisco has sparked extensive efforts to re-engineer the enzyme with the goal of enhancing agricultural productivity. To facilitate such efforts we analysed the formation of cyanobacterial form I Rubisco by in vitro reconstitution and cryo-electron microscopy. We show that RbcL subunit folding by the GroEL/GroES chaperonin is tightly coupled with assembly mediated by the chaperone RbcX2. RbcL monomers remain partially unstable and retain high affinity for GroEL until captured by RbcX2. As revealed by the structure of a RbcL8–(RbcX2)8 assembly intermediate, RbcX2 acts as a molecular staple in stabilizing the RbcL subunits as dimers and facilitates RbcL8 core assembly. Finally, addition of RbcS results in RbcX2 release and holoenzyme formation. Specific assembly chaperones may be required more generally in the formation of complex oligomeric structures when folding is closely coupled to assembly.

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Figure 1: GroEL/GroES-assisted folding of Syn6301-RbcL.
Figure 2: Reconstitution of Syn6301-RbcL 8 S 8 holoenzyme.
Figure 3: Cryo-electron microscopic structures of Rubisco assembly intermediates.
Figure 4: RbcX 2 –RbcL interactions.
Figure 5: The RbcL C-terminal domain becomes ordered when RbcX 2 is bound.
Figure 6: Model of GroEL/ES and RbcX 2 -assisted folding and assembly of Rubisco.

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  1. 1

    Gutteridge, S. & Gatenby, A. A. Rubisco synthesis, assembly, mechanism, and regulation. Plant Cell 7, 809–819 (1995)

    CAS  Article  Google Scholar 

  2. 2

    Andersson, I. & Taylor, T. C. Structural framework for catalysis and regulation in ribulose-1,5-bisphosphate carboxylase/oxygenase. Arch. Biochem. Biophys. 414, 130–140 (2003)

    CAS  Article  Google Scholar 

  3. 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. 4

    Newman, J., Branden, C. I. & Jones, T. A. Structure determination and refinement of ribulose 1,5-bisphosphate carboxylase/oxygenase from Synechococcus pcc6301. Acta Crystallogr. Sect D: Biol. Crystallogr. 49, 548–560 (1993)

    CAS  Article  Google Scholar 

  5. 5

    Newman, J. & Gutteridge, S. The X-ray structure of Synechococcus ribulose-bisphosphate carboxylase/oxygenase-activated quaternary complex at 2.2 Å resolution. J. Biol. Chem. 268, 25876–25886 (1993)

    CAS  PubMed  Google Scholar 

  6. 6

    Schneider, G., Lindqvist, Y. & Lundqvist, T. Crystallographic refinement and structure of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum at 1.7 Å resolution. J. Mol. Biol. 211, 989–1008 (1990)

    CAS  Article  Google Scholar 

  7. 7

    Andrews, T. J. & Lorimer, G. H. in The Biochemistry of plants: a comprehensive treatise (eds Hatch, M D. & Boardman, N K.) 10, 131–218 (1987)

    Google Scholar 

  8. 8

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

    CAS  Article  Google Scholar 

  9. 9

    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 

  10. 10

    Andrews, T. J. & Whitney, S. M. Manipulating ribulose bisphosphate carboxylase/oxygenase in the chloroplasts of higher plants. Arch. Biochem. Biophys. 414, 159–169 (2003)

    Article  Google Scholar 

  11. 11

    Spreitzer, R. J., Peddi, S. R. & Satagopan, S. Phylogenetic engineering at an interface between large and small subunits imparts land-plant kinetic properties to algal Rubisco. Proc. Natl Acad. Sci. USA 102, 17225–17230 (2005)

    ADS  CAS  Article  Google Scholar 

  12. 12

    Goloubinoff, P., Christeller, J. T., Gatenby, A. A. & Lorimer, G. H. Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfolded state depends on two chaperonin proteins and MgATP. Nature 342, 884–889 (1989)

    ADS  CAS  Article  Google Scholar 

  13. 13

    Brinker, A. et al. Dual function of protein confinement in chaperonin-assisted protein folding. Cell 107, 223–233 (2001)

    CAS  Article  Google Scholar 

  14. 14

    Hartl, F. U. & Hayer-Hartl, M. Converging concepts of protein folding in vitro and in vivo . Nature Struct. Mol. Biol. 16, 574–581 (2009)

    CAS  Article  Google Scholar 

  15. 15

    Whitney, S. M. & Sharwood, R. E. Construction of a tobacco master line to improve Rubisco engineering in chloroplasts. J. Exp. Bot. 59, 1909–1921 (2008)

    CAS  Article  Google Scholar 

  16. 16

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

    CAS  Article  Google Scholar 

  17. 17

    Tanaka, S., Sawaya, M. R., Kerfeld, C. A. & Yeates, T. O. Structure of the RuBisCO chaperone RbcX from Synechocystis sp PCC6803. Acta Crystallogr. Sect D. Biol. Crystallogr. 63, 1109–1112 (2007)

    CAS  Article  Google Scholar 

  18. 18

    Tarnawski, M., Gubernator, B., Kolesinski, P. & Szczepaniak, A. Heterologous expression and initial characterization of recombinant RbcX protein from Thermosynechococcus elongatus BP-1 and the role of RbcX in RuBisCO assembly. Acta Biochim. Pol. 55, 777–785 (2008)

    CAS  PubMed  Google Scholar 

  19. 19

    van der Vies, S. M., Bradley, D. & Gatenby, A. A. Assembly of cyanobacterial and higher plant ribulose bisphosphate carboxylase subunits into functional homologous and heterologous enzyme molecules in Escherichia coli . EMBO J. 5, 2439–2444 (1986)

    CAS  Article  Google Scholar 

  20. 20

    Andrews, T. J. Catalysis by cyanobacterial ribulose-bisphosphate carboxylase large subunits in the complete absence of small subunits. J. Biol. Chem. 263, 12213–12219 (1988)

    CAS  PubMed  Google Scholar 

  21. 21

    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)

    ADS  CAS  Article  Google Scholar 

  22. 22

    Li, L.-A. & Tabita, F. R. Maximum activity of recombinant ribulose 1,5-bisphosphate carboxylase/oxygenase of Anabaena sp. strain CA requires the product of the rbcX gene. J. Bacteriol. 179, 3793–3796 (1997)

    CAS  Article  Google Scholar 

  23. 23

    Onizuka, T. et al. The rbcX gene product promotes the production and assembly of ribulose-1,5-bisphosphate carboxylase/oxygenase of Synechococcus sp. PCC7002 in Escherichia coli . Plant Cell Physiol. 45, 1390–1395 (2004)

    CAS  Article  Google Scholar 

  24. 24

    Emlyn-Jones, D., Woodger, F. J., Price, G. D. & Whitney, S. M. RbcX can function as a Rubisco chaperonin, but is non-essential in Synechococcus PCC7942. Plant Cell Physiol. 47, 1630–1640 (2006)

    CAS  Article  Google Scholar 

  25. 25

    van der Vies, S. et al. Conformational states of ribulose bisphosphate carboxylase and their interaction with chaperonin 60. Biochem. 31, 3635–3644 (1992)

    CAS  Article  Google Scholar 

  26. 26

    Weissman, J. S. et al. Characterization of the active intermediate of a GroEL-GroES-mediated protein folding reaction. Cell 84, 481–490 (1996)

    CAS  Article  Google Scholar 

  27. 27

    Langer, T. et al. Chaperonin-mediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity. EMBO J. 11, 4757–4765 (1992)

    CAS  Article  Google Scholar 

  28. 28

    Martin, J., Mayhew, M., Langer, T. & Hartl, F. U. The reaction cycle of GroEL and GroES in chaperonin-assisted protein folding. Nature 366, 228–233 (1993)

    ADS  CAS  Article  Google Scholar 

  29. 29

    Martin, J. et al. Chaperonin-mediated protein folding at the surface of GroEL through a ‘molten globule’-like intermediate. Nature 352, 36–42 (1991)

    ADS  CAS  Article  Google Scholar 

  30. 30

    Thow, G., Zhu, G. H. & Spreitzer, R. J. Complementing substitutions within loop regions 2 and 3 of the α/β-barrel active site influence the CO2/O2 specificity of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase. Biochem. 33, 5109–5114 (1994)

    CAS  Article  Google Scholar 

  31. 31

    Tabita, F. R. et al. Function, structure, and evolution of the RubisCO-like proteins and their RubisCO homologs. Microbiol. Mol. Biol. Rev. 71, 576–599 (2007)

    CAS  Article  Google Scholar 

  32. 32

    Barraclough, R. & Ellis, R. J. Protein synthesis in chloroplasts. IX. Assembly of newly-synthesized large subunits into ribulose bisphosphate carboxylase in isolated intact pea chloroplasts. Biochim. Biophys. Acta 608, 19–31 (1980)

    CAS  Article  Google Scholar 

  33. 33

    Hayer-Hartl, M. K., Weber, F. & Hartl, F. U. Mechanism of chaperonin action: GroES binding and release can drive GroEL-mediated protein folding in the absence of ATP hydrolysis. EMBO J. 15, 6111–6121 (1996)

    CAS  Article  Google Scholar 

  34. 34

    Roessner, D. & Kulicke, W. M. On-line coupling of flow field-flow fractionation and multi-angle laser light scattering. J. Chromatogr. A 687, 249–258 (1994)

    CAS  Article  Google Scholar 

  35. 35

    Tang, Y. C. et al. Structural features of the GroEL-GroES nano-cage required for rapid folding of encapsulated protein. Cell 125, 903–914 (2006)

    CAS  Article  Google Scholar 

  36. 36

    Chin, J. W. & Schultz, P. G. In vivo photocrosslinking with unnatural amino acid mutagenesis. ChemBioChem 3, 1135–1137 (2002)

    CAS  Article  Google Scholar 

  37. 37

    Lakshmipathy, S. K. et al. Identification of nascent chain interaction sites on trigger factor. J. Biol. Chem. 282, 12186–12193 (2007)

    CAS  Article  Google Scholar 

  38. 38

    Weiner, M. P. et al. Site-directed mutagenesis of double-stranded DNA by the polymerase chain reaction. Gene 151, 119–123 (1994)

    CAS  Article  Google Scholar 

  39. 39

    Wiseman, T., Williston, S., Brandts, J. F. & Lin, L. N. Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem. 179, 131–137 (1989)

    CAS  Article  Google Scholar 

  40. 40

    Ludtke, S. J., Baldwin, P. R. & Chiu, W. EMAN: Semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999)

    CAS  Article  Google Scholar 

  41. 41

    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 

  42. 42

    Chin, J. W., Martin, A. B., King, D. S., Wang, L. & Schultz, P. G. Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli . Proc. Natl Acad. Sci. USA 99, 11020–11024 (2002)

    ADS  CAS  Article  Google Scholar 

  43. 43

    Ryu, Y. & Schultz, P. G. Efficient incorporation of unnatural amino acids into proteins in Escherichia coli . Nature Methods 3, 263–265 (2006)

    CAS  Article  Google Scholar 

  44. 44

    Leslie, A. G. W. Recent changes to the MOSFLM package for processing film and image plate data. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography 26, (1992)

  45. 45

    Evans, P. R. Scala. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography 33, 22–24 (1997)

    Google Scholar 

  46. 46

    Vagin, A. A. & Isupov, M. N. Spherically averaged phased translation function and its application to the search for molecules and fragments in electron-density maps. Acta Crystallogr. Sect D: Biol. Crystallogr. 57, 1451–1456 (2001)

    CAS  Article  Google Scholar 

  47. 47

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

    CAS  Article  Google Scholar 

  48. 48

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

    Article  Google Scholar 

  49. 49

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

    CAS  Article  Google Scholar 

  50. 50

    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 

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Assistance by K. Valer and J. Basquin in the MPIB crystallization facility, by C. Ungewickell in the Gene Center cryo-EM facility as well as by the staff at SLS-beamline X10SA-PX-II is acknowledged. We thank the Deutsche Forschungsgemeinschaft (SFB 594), the Ernst-Jung Foundation, the Körber Foundation, the European Union and the Center for Integrated Protein Science Munich (CIPSM) for financial support.

Author Contributions C.L. designed and executed most of the Rubisco reconstitution experiments and purified the Syn6301-RbcL8 core complexes with contributions from S.S., B.V.R. and K.V.R. The cryo-EM and three-dimensional image analysis were carried out by A.L.Y., as well as the interpretation and fitting of available crystal structures with contributions by O.B. and T.M. to data collection. A.S.-W. performed the crosslinking experiments, peptide binding assays and mutational analysis. M.H.-H. performed the FFF measurements. A.B. purified the Syn6301-RbcL8–AnaCA-(RbcX2)8 complexes and determined the crystal structure of AnaCA-RbcX2. R.B. supervised the design and interpretation of the cryo-EM analysis. M.H.-H. and F.U.H. supervised the design and interpretation of the biochemical experiments and wrote the manuscript with contributions from A.L.Y. and R.B.

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Correspondence to F. Ulrich Hartl or Roland Beckmann or Manajit Hayer-Hartl.

Additional information

The EM density maps are deposited to the 3D-EM database (EMD-1654, EMD-1655, EMD-1656) and the coordinates of the fitted RbcL8–(RbcX2)8 structure and the crystal structure of AnaCA-RbcX2 to the Protein Data Bank (2WVW and 3HYB, respectively).

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

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