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Molecular basis for the assembly of RuBisCO assisted by the chaperone Raf1

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Abstract

The folding and assembly of RuBisCO, the most abundant enzyme in nature, needs a series of chaperones, including the RuBisCO accumulation factor Raf1, which is highly conserved in cyanobacteria and plants. Here, we report the crystal structures of Raf1 from cyanobacteria Anabaena sp. PCC 7120 and its complex with RuBisCO large subunit RbcL. Structural analyses and biochemical assays reveal that each Raf1 dimer captures an RbcL dimer, with the C-terminal tail inserting into the catalytic pocket, and further mediates the assembly of RbcL dimers to form the octameric core of RuBisCO. Furthermore, the cryo-electron microscopy structures of the RbcL–Raf1–RbcS assembly intermediates enable us to see a dynamic assembly process from RbcL8Raf18 to the holoenzyme RbcL8RbcS8. In vitro assays also indicate that Raf1 can attenuate and reverse CcmM-mediated cyanobacterial RuBisCO condensation. Combined with previous findings, we propose a putative model for the assembly of cyanobacterial RuBisCO coordinated by the chaperone Raf1.

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Fig. 1: Crystal structures of Raf1 in apo- and RbcL-bound forms.
Fig. 2: The C-tail of Raf1 inserting into the catalytic pocket of RbcL and its contribution to the RbcL octamer assembly.
Fig. 3: Cryo-EM structures of RuBisCO assembly intermediates.
Fig. 4: Assembly of RuBisCO assisted by Raf1 and other chaperones.
Fig. 5: Raf1 antagonizes CcmM35-mediated condensation of RuBisCO.
Fig. 6: A putative model of chaperone-assisted assembly of cyanobacterial RuBisCO.

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Data availability

The structural factors and atomic coordinates of Raf1 and its complex with RbcL have been deposited at PDB (Raf1, 6KKN; L8F8, 6KKM). The cryo-EM structures of L8F8S8 and L8S4 have been deposited at PDB (L8F8S8, 6LRR; L8S4, 6LRS). The cryo-EM density maps of L8F8S8, L8S4, L8F8S4 and L8S8 have been deposited at the Electron Microscopy Data Bank (EMDB-0959–EMDB-0962, respectively). Source Data for Figs. 2 and 4–6 are provided with the paper.

References

  1. Bar-On, Y. M. & Milo, R. The global mass and average rate of RuBisCO. Proc. Natl Acad. Sci. USA 116, 4738–4743 (2019).

    Article  CAS  PubMed  Google Scholar 

  2. Ellis, R. J. The most abundant protein in the world. Trends Biochem. Sci. 4, 241–244 (1979).

    Article  CAS  Google Scholar 

  3. Bracher, A., Whitney, S. M., Hartl, F. U. & Hayer-Hartl, M. Biogenesis and metabolic maintenance of RuBisCO. Ann. Rev. Plant Biol. 68, 29–60 (2017).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  5. Whitney, S. M., Houtz, R. L. & Alonso, H. Advancing our understanding and capacity to engineer nature’s CO2-sequestering enzyme, RuBisCO. Plant Physiol. 155, 27–35 (2011).

    Article  CAS  PubMed  Google Scholar 

  6. Parry, M. A. et al. RuBisCO activity and regulation as targets for crop improvement. J. Exp. Bot. 64, 717–730 (2013).

    Article  CAS  PubMed  Google Scholar 

  7. Lin, M. T., Occhialini, A., Andralojc, P. J., Parry, M. A. & Hanson, M. R. A faster RuBisCO with potential to increase photosynthesis in crops. Nature 513, 547–550 (2014).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Erb, T. J. & Zarzycki, J. Biochemical and synthetic biology approaches to improve photosynthetic CO2-fixation. Curr. Opin. Chem. Biol. 34, 72–79 (2016).

    Article  CAS  PubMed  Google Scholar 

  9. Sharwood, R. E. Engineering chloroplasts to improve RuBisCO catalysis: prospects for translating improvements into food and fiber crops. New Phytol. 213, 494–510 (2017).

    Article  CAS  PubMed  Google Scholar 

  10. Hauser, T., Popilka, L., Hartl, F. U. & Hayer-Hartl, M. Role of auxiliary proteins in RuBisCO biogenesis and function. Nat. Plants 1, 15065 (2015).

    Article  CAS  PubMed  Google Scholar 

  11. Wilson, R. H. & Hayer-Hartl, M. Complex chaperone dependence of RuBisCO biogenesis. Biochemistry 57, 3210–3216 (2018).

    Article  CAS  PubMed  Google Scholar 

  12. 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  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  15. Bracher, A., Starling-Windhof, A., Hartl, F. U. & Hayer-Hartl, M. Crystal structure of a chaperone-bound assembly intermediate of form I RuBisCO. Nat. Struct. Mol. Biol. 18, 875–880 (2011).

    Article  CAS  PubMed  Google Scholar 

  16. Hauser, T. et al. Structure and mechanism of the RuBisCO-assembly chaperone Raf1. Nat. Struct. Mol. Biol. 22, 720–728 (2015).

    Article  CAS  PubMed  Google Scholar 

  17. 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 

  18. Kolesinski, P., Belusiak, I., Czarnocki-Cieciura, M. & Szczepaniak, A. RuBisCO accumulation factor 1 from Thermosynechococcus elongatus participates in the final stages of ribulose-1,5-bisphosphate carboxylase/oxygenase assembly in Escherichia coli cells and in vitro. FEBS J. 281, 3920–3932 (2014).

    Article  CAS  PubMed  Google Scholar 

  19. Kerfeld, C. A. & Melnicki, M. R. Assembly, function and evolution of cyanobacterial carboxysomes. Curr. Opin. Plant Biol. 31, 66–75 (2016).

    Article  CAS  PubMed  Google Scholar 

  20. Long, B. M., Tucker, L., Badger, M. R. & Price, G. D. Functional cyanobacterial beta-carboxysomes have an absolute requirement for both long and short forms of the CcmM protein. Plant Physiol. 153, 285–293 (2010).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Turmo, A., Gonzalez-Esquer, C. R. & Kerfeld, C. A. Carboxysomes: metabolic modules for CO2 fixation. FEMS Microbiol. Lett. 364, fnx176 (2017).

  22. Aigner, H. et al. Plant RuBisCO assembly in E. coli with five chloroplast chaperones including BSD2. Science 358, 1272–1278 (2017).

    Article  CAS  PubMed  Google Scholar 

  23. Feiz, L. et al. Ribulose-1,5-bis-phosphate carboxylase/oxygenase accumulation factor 1 is required for holoenzyme assembly in maize. Plant Cell 24, 3435–3446 (2012).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. Salesse-Smith, C. E. et al. Overexpression of RuBisCO subunits with RAF1 increases RuBisCO content in maize. Nat. Plants 4, 802–810 (2018).

    Article  CAS  PubMed  Google Scholar 

  25. Kolesinski, P., Rydzy, M. & Szczepaniak, A. Is RAF1 protein from Synechocystis sp. PCC 6803 really needed in the cyanobacterial RuBisCO assembly process? Photosynth. Res. 132, 135–148 (2017).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Cleland, W. W., Andrews, T. J., Gutteridge, S., Hartman, F. C. & Lorimer, G. H. Mechanism of RuBisCO: the carbamate as general base. Chem. Rev. 98, 549–562 (1998).

    Article  CAS  PubMed  Google Scholar 

  27. Newman, J., Branden, C. I. & Jones, T. A. Structure determination and refinement of ribulose 1,5-bisphosphate carboxylase/oxygenase from Synechococcus PCC 6301. Acta Crystallogr. D 49, 548–560 (1993).

    Article  CAS  PubMed  Google Scholar 

  28. Duff, A. P., Andrews, T. J. & Curmi, P. M. The transition between the open and closed states of RuBisCO is triggered by the inter-phosphate distance of the bound bisphosphate. J. Mol. Biol. 298, 903–916 (2000).

    Article  CAS  PubMed  Google Scholar 

  29. Whitney, S. M., Birch, R., Kelso, C., Beck, J. L. & Kapralov, M. V. Improving recombinant RuBisCO biogenesis, plant photosynthesis and growth by coexpressing its ancillary RAF1 chaperone. Proc. Natl Acad. Sci. USA 112, 3564–3569 (2015).

    Article  CAS  PubMed  Google Scholar 

  30. Taylor, T. C. & Andersson, I. Structure of a product complex of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase. Biochemistry 36, 4041–4046 (1997).

    Article  CAS  PubMed  Google Scholar 

  31. Taylor, T. C. & Andersson, I. The structure of the complex between RuBisCO and its natural substrate ribulose 1,5-bisphosphate. J. Mol. Biol. 265, 432–444 (1997).

    Article  CAS  PubMed  Google Scholar 

  32. Hartman, F. C. & Harpel, M. R. Structure, function, regulation, and assembly of d-ribulose-1,5-bisphosphate carboxylase/oxygenase. Annu. Rev. Biochem. 63, 197–234 (1994).

    Article  CAS  PubMed  Google Scholar 

  33. Alonso, H., Blayney, M. J., Beck, J. L. & Whitney, S. M. Substrate-induced assembly of Methanococcoides burtonii d-ribulose-1,5-bisphosphate carboxylase/oxygenase dimers into decamers. J. Biol. Chem. 284, 33876–33882 (2009).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Hayer-Hartl, M. From chaperonins to RuBisCO assembly and metabolic repair. Protein Sci. 26, 2324–2333 (2017).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Vitlin Gruber, A. & Feiz, L. RuBisCO assembly in the chloroplast. Front. Mol. Biosci. 5, 24 (2018).

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Wang, H. et al. RuBisCO condensate formation by CcmM in β-carboxysome biogenesis. Nature 566, 131–135 (2019).

    Article  CAS  PubMed  Google Scholar 

  37. Ryan, P. et al. The small RbcS-like domains of the beta-carboxysome structural protein CcmM bind RuBisCO at a site distinct from that binding the RbcS subunit. J. Biol. Chem. 294, 2593–2603 (2019).

    Article  CAS  PubMed  Google Scholar 

  38. Joshi, J., Mueller-Cajar, O., Tsai, Y. C., Hartl, F. U. & Hayer-Hartl, M. Role of small subunit in mediating assembly of red-type form I RuBisCO. J. Biol. Chem. 290, 1066–1074 (2015).

    Article  CAS  PubMed  Google Scholar 

  39. 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 PCC 7942. Plant Cell Physiol. 47, 1630–1640 (2006).

    Article  CAS  PubMed  Google Scholar 

  40. Wang, Q. S. et al. The macromolecular crystallography beamline of SSRF. Nucl. Sci. Tech. 26, 12–17 (2015).

    Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  42. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  PubMed  Google Scholar 

  43. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  45. Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355–367 (2011).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  47. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

    Article  CAS  PubMed  Google Scholar 

  48. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  49. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  51. 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 

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

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank the staff at the Shanghai Synchrotron Radiation Facility (SSRF) for X-ray diffraction data collection; L. Chen and B. Zhu for technical support with cryo-EM data collection at the Center for Biological Imaging at the Institute of Biophysics (IBP), Chinese Academy of Sciences; and the staff at the Core Facility Center for Life Sciences at University of Science and Technology of China for technical assistance. This research was supported by the National Natural Science Foundation of China (http://www.nsfc.gov.cn; grant numbers 31630001 and 31621002), the Strategic Priority Research Program of the Chinese Academy of Sciences (http://www.cas.cn; grant numbers XDA24020302 and XDB37020301) and the Ministry of Science and Technology of China (http://www.most.gov.cn; project number 2016YFA0400900). Y.-L.J. thanks the Youth Innovation Promotion Association of Chinese Academy of Sciences for their support.

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  1. These authors contributed equally: Ling-Yun Xia, Yong-Liang Jiang, Wen-Wen Kong.

Authors and Affiliations

  1. Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, China

    Ling-Yun Xia, Yong-Liang Jiang, Wen-Wen Kong, Hui Sun, Wei-Fang Li, Yuxing Chen & Cong-Zhao Zhou

  2. Innovation Academy for Seed Design, Chinese Academy of Sciences, Hefei, China

    Ling-Yun Xia, Yong-Liang Jiang, Wen-Wen Kong & Cong-Zhao Zhou

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Contributions

C.-Z.Z., Y.-L.J. and Y.C. conceived, designed and supervised the project. Y.-L.J., C.-Z.Z., Y.C., W.-F.L. and L.-Y.X. analysed the data. Y.-L.J. and C.-Z.Z. wrote the manuscript. L.-Y.X., W.-W.K. and H.S. performed the molecular cloning, protein expression and purification. L.-Y.X. and W.-W.K. performed protein crystallization and optimization. Y.-L.J. and L.-Y.X. conducted the X-ray and cryo-EM data collection, structure determination and model refinement. L.-Y.X. and W.-W.K. performed the biochemical assays. All of the authors discussed the data and read the manuscript.

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Correspondence to Yong-Liang Jiang, Yuxing Chen or Cong-Zhao Zhou.

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Peer review information: Nature Plants thanks Oliver Martin Mueller-Cajar, Spencer Whitney and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Three interfaces between two subunits of Raf1 dimer.

The two Raf1 subunits (orange and cyan) are shown as semi-transparent cartoons, whereas the interacting residues are shown as sticks, with polar interactions indicated as dashed lines. a, The interface between two Raf1β domains. c, The interface between the hydrophobic linker of one subunit and Raf1β of the symmetric subunit. c, The interface between Raf1α of one subunit and Raf1β of the symmetric subunit.

Extended Data Fig. 2 Multiple-sequence alignment of Raf1 homologs in cyanobacteria and plants.

Amino acid sequences of Raf1 homologs from cyanobacteria and plants are aligned using MultAlin (http://multalin.toulouse.inra.fr/multalin/). The secondary structural elements of Anabaena sp. PCC 7120 Raf1 are labeled at the top. Three interfaces (interface-I, II, III) between Raf1 and RbcL are labeled at the bottom, with the interacting residues at the three interfaces indicated by pink triangles, cyan squares, and red circles, respectively. Residues in the three interfaces between the subunits of Raf1 dimer are labeled with orange, yellow and blue stars, respectively. The linker region connecting the two Raf1 domains is marked by a pink line on the top of the sequences. The NCBI accession codes for the sequences are: Anabaena sp. PCC 7120, WP_010999374; Oscillatoriales cyanobacterium, TAF57237; Synechocystis sp. PCC 6803, WP_010873864; Synechococcus elongatus PCC 7942, WP_011377752; Thermosynechococcus elongatus BP-1, WP_011057603; Limnoraphis robusta, WP_046279487; Arabidopsis thaliana, NP_198202; Cajanus cajan, XP_020203342; Spinacia oleracea, XP_021866119; Helianthus annuus, XP_021973841; Zea mays, NP_001140763; Marchantia polymorpha, PTQ50472.

Extended Data Fig. 3 The complementary electrostatic surfaces presentation of the RbcL dimer and Raf1, showing the areas at interface-II and III.

The interacting areas on RbcL and Raf1 are highlighted as black dashed lines. The interface-II and III show a substantial complementarity in shape and charge between RbcL and Raf1α.

Extended Data Fig. 4 Superposition of Anabaena sp. PCC 7120 Raf1 in apo and RbcL-bound forms.

Superposition of individual (a) Raf1α and (b) Raf1β domains in apo (blue) and RbcL-bound forms (cyan). c, Structural comparison of Raf1 dimer in the apo and RbcL-bound forms, shown in two orientations rotated by 90°. The Raf1β domains were aligned together, in which Raf1α domains rotates against the swapped Raf1β dimer by ~75°.

Extended Data Fig. 5 Comparison of the ‘loop 6’ in the structures of L8F8 and RuBisCO holoenzyme L8S8.

The Raf1 C-tail partially occupies the space, which is held by the ‘loop 6’ in the RuBisCO.

Extended Data Fig. 6 Native- and SDS-PAGE analysis of altered RbcL-containing complex formation upon mutations of Raf1 C-tail.

a, Native-PAGE analysis of RbcL-Raf1 complex with addition of RbcS at various concentrations (0, 4, 8 and 20 µM, with the molar ratio of 0, 0.5, 1 and 2.5 to RbcL, respectively). WT, CH3 and ∆C8 represent the wild-type Raf1 or Raf1 mutants, in which CH3 stands for extension of three histidine residues at the C-terminus whereas ∆C8 represents the truncation of the C-terminal eight residues. The numerically labeled bands in panel a are cut off for further analysis by SDS-PAGE in the lanes of (b) WT, (c) CH3 and (d) ∆C8, respectively.

Source data

Extended Data Fig. 7 Raf1α and RbcS share a largely overlapped binding regions on RbcL.

The RbcL structures are shown as surface. The binding regions of Raf1α and RbcS on RbcL are circled by dashed lines in cyan and gold, respectively. The shared binding residues on RbcL are shown in sticks and labeled.

Extended Data Fig. 8 Comparison of the RbcS-binding residues of RbcL in the structures of L8F8 and the holoenzyme L8S8.

RbcS-binding residues are shown as blue and gray sticks for L8F8 and L8S8, respectively.

Extended Data Fig. 9 Raf1α and SSUL1 possess a slightly overlapped binding region on RbcL.

Superposition of L8F8 onto the complex of L8S8-SSUL1 (PDB: 6HBC). The RbcL structures are shown as surface, whereas the binding regions of Raf1 and SSUL1 are circled by the cyan and violet dashed lines, respectively.

Extended Data Fig. 10 The C-terminus of RbcL interacts with Raf1.

The RbcL and Raf1 structures are shown as electrostatic surface. The C-terminal residues of RbcL are shown as pink sticks, whereas Raf1 residues interacting with the C-terminus of RbcL are shown as cyan and orange sticks for the two subunits.

Supplementary information

Supplementary Information

Supplementary Figs. 1–10 and Tables 1–4.

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Source data

Source Data Fig. 2

The full-length unprocessed western blot of Fig. 2e and the full-length gel of Fig. 2f.

Source Data Fig. 4

The full-length unprocessed gels of Fig. 4a.

Source Data Fig. 4

The statistics source data of Fig. 4b.

Source Data Fig. 5

The statistics source data of Fig. 5a and Fig. 5c.

Source Data Extended Data Fig. 6

The full-length unprocessed gels of Extended Data Fig. 6.

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Xia, LY., Jiang, YL., Kong, WW. et al. Molecular basis for the assembly of RuBisCO assisted by the chaperone Raf1. Nat. Plants 6, 708–717 (2020). https://doi.org/10.1038/s41477-020-0665-8

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