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Structure and mechanism of the Rubisco-assembly chaperone Raf1

Nature Structural & Molecular Biology volume 22, pages 720–728 (2015)Cite this article

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Abstract

Biogenesis of the photosynthetic enzyme Rubisco, a complex of eight large (RbcL) and eight small (RbcS) subunits, requires assembly chaperones. Here we analyzed the role of Rubisco accumulation factor1 (Raf1), a dimer of ∼40-kDa subunits. We find that Raf1 from Synechococcus elongatus acts downstream of chaperonin-assisted RbcL folding by stabilizing RbcL antiparallel dimers for assembly into RbcL8 complexes with four Raf1 dimers bound. Raf1 displacement by RbcS results in holoenzyme formation. Crystal structures show that Raf1 from Arabidopsis thaliana consists of a β-sheet dimerization domain and a flexibly linked α-helical domain. Chemical cross-linking and EM reconstruction indicate that the β-domains bind along the equator of each RbcL2 unit, and the α-helical domains embrace the top and bottom edges of RbcL2. Raf1 fulfills a role similar to that of the assembly chaperone RbcX, thus suggesting that functionally redundant factors ensure efficient Rubisco biogenesis.

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Figure 1: Raf1-mediated assembly of S. elongatus Rubisco.
Figure 2: Interaction of Raf1 with preformed RbcL8 complexes.
Figure 3: Crystal structures of Raf1 domains.
Figure 4: Structure-based mutational analysis of Raf1.
Figure 5: Probing the RbcL–Raf1 complex by chemical cross-linking.
Figure 6: Negative-stain EM and 3D reconstructions of RbcL8–Raf14 complex.
Figure 7: Model of Raf1-mediated Rubisco assembly.

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References

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

    CAS  PubMed  Google Scholar 

  2. 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).

    CAS  PubMed  Google Scholar 

  3. Ellis, R.J. Biochemistry: tackling unintelligent design. Nature 463, 164–165 (2010).

    CAS  PubMed  Google Scholar 

  4. Sharkey, T.D. Estimating the rate of photorespiration. Physiol. Plant. 73, 147–152 (1988).

    CAS  Google Scholar 

  5. Portis, A.R. & Parry, M.A.J. Discoveries in Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase): a historical perspective. Photosynth. Res. 94, 121–143 (2007).

    CAS  PubMed  Google Scholar 

  6. Peterhansel, C., Niessen, M. & Kebeish, R.M. Metabolic engineering towards the enhancement of photosynthesis. Photochem. Photobiol. 84, 1317–1323 (2008).

    CAS  PubMed  Google Scholar 

  7. Maurino, V.G. & Peterhansel, C. Photorespiration: current status and approaches for metabolic engineering. Curr. Opin. Plant Biol. 13, 249–256 (2010).

    PubMed  Google Scholar 

  8. 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).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  10. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Durão, P. et al. Opposing effects of folding and assembly chaperones on evolvability of Rubisco. Nat. Chem. Biol. 11, 148–155 (2015).

    PubMed  Google Scholar 

  12. 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).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  15. Vitlin Gruber, A., Nisemblat, S., Azem, A. & Weiss, C. The complexity of chloroplast chaperonins. Trends Plant Sci. 18, 688–694 (2013).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  17. 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  PubMed  PubMed Central  Google Scholar 

  18. 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 

  19. 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).

    CAS  PubMed  Google Scholar 

  20. 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  PubMed  PubMed Central  Google Scholar 

  21. 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  PubMed  Google Scholar 

  22. 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).

    CAS  PubMed  Google Scholar 

  23. 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  PubMed  Google Scholar 

  24. Feiz, L. et al. Ribulose-1,5-bis-phosphate carboxylase/oxygenase accumulation factor1 is required for holoenzyme assembly in maize. Plant Cell 24, 3435–3446 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 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).

    CAS  PubMed  Google Scholar 

  26. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Leitner, A. et al. The molecular architecture of the eukaryotic chaperonin TRiC/CCT. Structure 20, 814–825 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Newman, J., Bränden, C.I. & Jones, T.A. Structure determination and refinement of ribulose 1,5-bisphosphate carboxylase/oxygenase from Synechococcus PCC6301. Acta Crystallogr. D Biol. Crystallogr. 49, 548–560 (1993).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  30. Chari, A. & Fischer, U. Cellular strategies for the assembly of molecular machines. Trends Biochem. Sci. 35, 676–683 (2010).

    CAS  PubMed  Google Scholar 

  31. Ellis, R.J. Assembly chaperones: a perspective. Phil. Trans. R. Soc. Lond. B 368, 20110398 (2013).

    Google Scholar 

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

    CAS  PubMed  Google Scholar 

  33. Kolesinski, P. et al. Insights into eukaryotic Rubisco assembly: crystal structures of RbcX chaperones from Arabidopsis thaliana. Biochim. Biophys. Acta 1830, 2899–2906 (2013).

    CAS  PubMed  Google Scholar 

  34. Wheatley, N.M., Sundberg, C.D., Gidaniyan, S.D., Cascio, D. & Yeates, T.O. Structure and identification of a pterin dehydratase-like protein as a ribulose-bisphosphate carboxylase/oxygenase (RuBisCO) assembly factor in the alpha-carboxysome. J. Biol. Chem. 289, 7973–7981 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Feiz, L. et al. RAF2: a novel Rubisco biogenesis factor in maize. Plant J. 80, 862–869 (2014).

    CAS  PubMed  Google Scholar 

  36. Hohmann-Marriott, M.F. & Blankenship, R.E. Evolution of photosynthesis. Annu. Rev. Plant Biol. 62, 515–548 (2011).

    CAS  PubMed  Google Scholar 

  37. Kerner, M.J. et al. Proteome-wide analysis of chaperonin-dependent protein folding in Escherichia coli. Cell 122, 209–220 (2005).

    CAS  PubMed  Google Scholar 

  38. Georgescauld, F. et al. GroEL/ES chaperonin modulates the mechanism and accelerates the rate of TIM-barrel domain folding. Cell 157, 922–934 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 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  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  42. Wyatt, P.J. Light scattering and the absolute characterization of macromolecules. Anal. Chim. Acta 272, 1–40 (1993).

    CAS  Google Scholar 

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

    PubMed  Google Scholar 

  44. Scheres, S.H. Semi-automated selection of cryo-EM particles in RELION-1.3. J. Struct. Biol. 189, 114–122 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 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  PubMed  Google Scholar 

  46. Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858 (1996).

    CAS  PubMed  Google Scholar 

  47. Rappsilber, J., Ishihama, Y. & Mann, M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663–670 (2003).

    CAS  PubMed  Google Scholar 

  48. Götze, M. et al. Automated assignment of MS/MS cleavable crosslinks in protein 3D-structure analysis. J. Am. Soc. Mass Spectrom. 26, 83–97 (2015).

    PubMed  Google Scholar 

  49. Vachette, P., Koch, M.H. & Svergun, D.I. Looking behind the beamstop: X-ray solution scattering studies of structure and conformational changes of biological macromolecules. Methods Enzymol. 374, 584–615 (2003).

    CAS  PubMed  Google Scholar 

  50. Konarev, P.V., Volkov, V.V., Sokolova, A.V., Koch, M.H.J. & Svergun, D.I. PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J. Appl. Crystallogr. 36, 1277–1282 (2003).

    CAS  Google Scholar 

  51. Svergun, D.I. Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J. Appl. Crystallogr. 25, 495–503 (1992).

    CAS  Google Scholar 

  52. Bernadó, P., Mylonas, E., Petoukhov, M.V., Blackledge, M. & Svergun, D.I. Structural characterization of flexible proteins using small-angle X-ray scattering. J. Am. Chem. Soc. 129, 5656–5664 (2007).

    PubMed  Google Scholar 

  53. Petoukhov, M.V. et al. New developments in the program package for small-angle scattering data analysis. J. Appl. Crystallogr. 45, 342–350 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    PubMed  Google Scholar 

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

    Google Scholar 

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

    Google Scholar 

  58. Sheldrick, G.M. Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Crystallogr. D Biol. Crystallogr. 66, 479–485 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Pape, T. & Schneider, T.R. HKL2MAP: a graphical user interface for phasing with SHELX programs. J. Appl. Crystallogr. 37, 843–844 (2004).

    CAS  Google Scholar 

  60. Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. Automated structure solution with autoSHARP. Methods Mol. Biol. 364, 215–230 (2007).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    PubMed  Google Scholar 

  63. Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D Biol. Crystallogr. 62, 1002–1011 (2006).

    PubMed  Google Scholar 

  64. 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. D Biol. Crystallogr. 57, 1451–1456 (2001).

    CAS  PubMed  Google Scholar 

  65. 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  PubMed  Google Scholar 

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

  67. Afonine, P.V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  70. Larkin, M.A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  72. Cole, C., Barber, J.D. & Barton, G.J. The Jpred 3 secondary structure prediction server. Nucleic Acids Res. 36, W197–W201 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

A. thaliana cDNA was a kind gift from B. Bölter (Ludwig-Maximilians-Universität München), and S. elongatus PCC7942 DNA was a kind gift from M. Hagemann (Universität Rostock). We thank A. Jungclaus for assistance with protein purification and R. Körner for performing the MS on the cross-linked samples. We also thank A. Sinz (Martin Luther University Halle-Wittenberg) for making available StavroX software for the cross-linking MS analysis. Assistance by K. Valer and J. Basquin at the Max Planck Institute of Biochemistry (MPIB) crystallization facility, as well as the staff at Swiss Light Source beamlines X10SA-PX-II and X06DA-PXIII, is gratefully acknowledged. SAXS data were collected at European Synchrotron Radiation Facility beamline BM29 with the assistance of A. Round. Cryo-EM analysis was performed in the Department of W. Baumeister at MPIB. The authors thank A. Kahraman (Universität Zürich) for helpful discussions regarding the interpretation of cross-linking data and D. Balchin and L. Popilka for critically reading the manuscript. P.W. is supported by an Emmy Noether grant of the German Research council (WE 4628/1).

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Authors and Affiliations

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

    Thomas Hauser, Javaid Y Bhat, Goran Miličić, F Ulrich Hartl, Andreas Bracher & Manajit Hayer-Hartl

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

    Petra Wendler

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Contributions

T.H. performed the biochemical and functional analysis of Raf1 and obtained the Raf1-domain crystals; A.B. solved the crystal structures; J.Y.B. performed the native MS and analyzed the cross-linking MS data; M.H.-H. did the SEC-MALS measurements; and G.M. and P.W. performed the cryo-EM analysis and reconstruction. M.H.-H., F.U.H. and A.B. supervised the experimental design and data interpretation, and wrote the manuscript with contributions from all authors.

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Correspondence to Andreas Bracher or Manajit Hayer-Hartl.

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Integrated supplementary information

Supplementary Figure 1 Alignment of Raf1 sequences.

Amino acid sequences of a representative set of Raf1 homologs were aligned using the EBI Clustal-Ω server. Secondary structure elements for Raf1.2 from Arabidopsis thaliana are indicated above the sequences. The Raf1 domain structure is indicated by purple and orange coloring of secondary structure elements in the Raf1α and Raf1β domains, respectively. The sequences from plants (1), green algae (2) and cyanobacteria (3) are grouped separately. Similar residues are shown in red and identical residues in white on a red background. Blue frames indicate homologous regions. The consensus sequence is shown at the bottom. The chloroplast signal sequences are not shown. Asterisks below the sequence indicate mutations in Syn7942-Raf1 analyzed in this study (Fig. 4). The Uniprot accession codes for the sequences are: Q9SR19, Arabidopsis thaliana Raf1.2; Q9LKR8, Arabidopsis thaliana Raf1.1; B4FR29, Zea mays; I0YJW5, Coccomyxa subellipsoidea C-169; E1ZGR5, Chlorella variabilis; Cre06.g308450.t1.2, Chlamydomonas reinhardtii; Q00S02, Ostreococcus tauri; C1FI81, Micromonas sp. (strain RCC299 / NOUM17); B4VSU9, Coleofasciculus chthonoplastes PCC7420; Q31Q05, Synechococcus elongatus PCC7942; Q5N472, Synechococcus elongatus PCC6301; B1XK11, Synechococcus sp. PCC7002.

Supplementary Figure 2 Functional analysis of Raf1 homologs.

(a) Purified full-length Raf1 proteins and the respective α- and b-domains of Syn7942, Syn7002 and A. thaliana. AtRaf1.1/1.2 is a complex of AtRaf1.1 and AtRaf1.2 produced from a biscistronic plasmid. (b) Native-PAGE analysis of Rubisco reconstitution reactions as in Fig. 1b, containing the Raf1 proteins indicated. (c) Rubisco activities in reactions shown in (b) after supplementation with RbcS as described in Fig. 1d. Error bars, s.d. (n = 3 independent experiments). (d) Displacement of Raf1 from RbcL8 by RbcS. Purified S. elongatus RbcL8 was incubated with Syn7942-Raf1 as in Fig. 2a, followed by addition of RbcS (5 μM) and analysis by native-PAGE and immunoblotting with anti-RbcL (left) and anti-Raf1 (right). S. elongatus RbcL8 and RbcL8S8 were used as standards. (e) Stoichiometry of RbcL and Raf1 in RbcL* complexes. RbcL* was excised from native-PAGE and separated by SDS-PAGE followed by Coomassie staining and quantitation by densitometry. Top, molar ratios of RbcL and Raf1 standards as quantified by extinction coefficients. Bottom, ratios of RbcL to Raf1 stain intensities are indicated as averages ±S.D from four measurements. The molar ratio of RbcL to Raf1 in RbcL* is close 1:1. Shown is a representative Coomassie stained gel. (f) Dependence of Rubisco assembly on Raf1 concentration. Reconstitution reactions were performed as in Fig. 1e at increasing concentrations of Raf1 and the Rubisco activities obtained after 60 min are indicated as percentage of control. Error bars, s.d. (n = 3 independent experiments).

Supplementary Figure 3 SEC-MALS and SAXS analysis of Raf1 proteins.

(a) SEC-MALS analysis of purified Raf1 domains from Syn7942, Syn7002 and A. thaliana. Data showing measurements for ~30 μg of the respective proteins. Horizontal lines across the peaks indicate molar mass and homogeneity of the sample. Calculated molar masses and hydrodynamic radii are indicated. (b) Representative X-ray scattering curves of AtRaf1.2 (red), AtRaf1.2α (blue) and AtRaf1.2β (green) and Syn7942-Raf1 (black). The curves represent background-corrected averages of ten measurements. The GNOM-fitted51 curves are overlaid. (c) Density distributions for AtRaf1.2 (red), AtRaf1.2α (blue), AtRaf1.2β (green) and Syn7942-Raf1 (black) calculated with GNOM. AtRaf1.2α and AtRaf1.2β appear rod-shaped and globular, respectively. The curves for AtRaf1.2 and Syn7942-Raf1 suggests flexibly linked domains.(d) Parameters from SAXS data analysis. Radii of gyration were determined using the Guinier approximation. Scattering curves were fitted with GNOM. (e) Ensemble model for the AtRaf1.2 dimer. Two perpendicular views are shown. The backbones are represented as coils. A subset of five models matching the experimental scattering curve (Chi value 3.978) was picked from a library of 10,000 conformations by a genetic algorithm implemented in the program EOM 2.052,53. The position of the dimeric β-domain (orange) was fixed at the dyad symmetry axis. The α-domains are represented in purple; the flexible termini and inter-domain linkers are shown in gray.

Supplementary Figure 4 Functional analysis of Raf1 homologs.

(a) Native-PAGE analysis of Rubisco reconstitution reactions as in Fig. 1b, containing purified full-length Raf1 and the α- and β-domains from Syn7942 and A. thaliana. RbcS was present when indicated. (b) Rubisco activities in reactions shown in (a) after supplementation with RbcS when absent, as described in Fig. 1d. Error bars, s.d. (n = 3 independent experiments).

Supplementary Figure 5 Crystal structures of AtRaf1.2 domains.

(a,b) Experimental electron density maps for AtRaf1.2α and AtRaf1.2β. Representative regions are shown. The meshwork represents the isocontour surface at 1.0 σ level. The nominal resolutions of the AtRaf1.2α Pt-SIRAS and AtRaf1.2β Hg-SIRAS maps are 2.75 and 3.0 Å, respectively. Panel B shows a contact between two AtRaf1.2β dimers. (c) Surface properties of AtRaf1.2a. The same views as in Fig. 3c are shown. Positively and negatively charged groups are shown in blue and red, respectively. Yellow color signifies hydrophobic sidechains. (d) Superposition of three crystallographically independent copies of the AtRaf1.2β dimer. The models are represented as Cα traces. The orientation corresponds to the top-view in Fig. 3d.(e) Domain swapping in the P212121 crystal lattice of AtRaf1.2β. In this lattice the long βF-βG connecting loops reach across between adjacent dimers, making contacts to a hydrophobic pit. In the C2 crystal form, the hydrophobic residues at the loop apex fold back onto the respective hydrophobic area of the same chain, realizing an analogous intramolecular contact. Outside of the crystal lattice the conformation observed in the C2 crystal form should be strongly favored. (f) Topology of the secondary structure in the AtRaf1.2β dimer. α-Helices and β-strands are represented by cylinders and arrows, using the same color scheme as in the main text. The monomer shown in orange differs from the second by insertion of helix 12. (g) Features of the AtRaf1.2β dimer interface. On the left, the surface properties of the area facing the RbcL dimer are show using the same representation as in in panel c. On the right, one monomer is shown as backbone ribbon, the other in surface representation to reveal the AtRaf1.2β dimer interface. Yellow color signifies hydrophobic sidechains.

Supplementary Figure 6 Cross-linking coupled to mass spectrometry (CXMS).

(a) Structure of the bifunctional crosslinker disuccinimidylsuberate (DSS). The crosslinker is a 1:1 mixture of unlabeled (light; H12) and deuterium labeled (heavy; D12) compounds with a mass difference of 12.0753 Da. (b) Workflow for analysis of crosslinked protein bands marked and numbered in (c) by in-gel trypsin digestion, followed by LC–MS. (c) Crosslinking products of individual proteins S. elongatus RbcL8, Syn7942-Raf1 and Syn7002-Raf1. The purified proteins (1.25 μM RbcL8 and 10 μM of the respective Raf1 proteins) were incubated with H12:D12–DSS (2 mM) for 30 min at 25°C, followed by quenching of the crosslinking reaction with NH4HCO3 (150 mM) and analysis by SDS-PAGE. (d) Crosslinking products of RbcL8 (1.25 μM) with Syn7942-Raf1 or Syn7002-Raf1 (10 μM each). Boxed areas were analyzed as in (b). (e) Representative MS/MS spectra for the crosslinks RbcL–RbcL (Lys15–Lys460), Raf1β–RbcL (Lys344–Lys336) and Raf1β–Raf1α (Lys344–Lys188).

Supplementary Figure 7 Negative-stain EM analysis.

(a) Fourier Shell Correlation (FSC) curves of S. elongatus (Se) RbcL8–Raf14, crosslinked SeRbcL8–Raf14, and T. elongatus (Te) RbcL8–Raf14 as determined by gold standard FSC procedure in RELION-1.3. The resolution of the maps estimated by FSC with 0.5 and 0.143 correlation cut-off and no masking are given. (b) Comparison of the RbcL8–Raf14 models derived from CXMS distance restraints (Fig. 5g,h) and EM reconstruction (Fig. 6f,h,j) (assuming the C-terminal 65 residues of RbcL are structured). The backbones are represented by Cα traces. Raf1 and RbcL in the CXMS model are shown in magenta and white, respectively. Raf1 and RbcL in the EM reconstruction are shown in cyan and gray, respectively. (c) Rigid body domain fitting of SeRaf1α- and β-domains and RbcL8 missing the C-terminal 65 amino acids into the 3D reconstruction of SeRbcL8–Raf14. RbcL subunits in gray and black; Raf1α in purple and Raf1β in orange. Side- and top-views are shown. (d,e) Negative stain electron micrograph of crosslinked SeRbcL8–Raf14 (d) and of TeRbcL8–Raf14 (e). Exemplary class averages of the respective complexes obtained from 2D classification in RELION-1.3 are shown in the insets.

Supplementary Figure 8 Characterization of the RbcL–Raf1 complex of the thermophilic cyanobacterium T. elongatus.

T. elongatus RbcL and Raf1 proteins were coexpressed in E. coli and purified as a high molecular weight complex. (a) SEC-MALS analysis of RbcL-Raf1 complex in solution (~40 μg). The horizontal line across the peak indicates the calculated molar mass. Note that the sample contained a small amount of aggregated protein which leads to a higher average molar mass (~828 kDa) than expected for the RbcL8–Raf14 complex (~740 kDa). (b) nano-ESI native MS spectra of RbcL–Raf1 complex (~8 μM), Symbols indicate the charge state distributions with the charge states shown for some peaks; the calculated mass around the m/z values of the respective protein complexes is indicated. S.D. refers to the accuracy of mass values calculated from the different m/z peaks. The theoretical masses for RbcL8–Raf14 and RbcL2 are 741628.8 Da and 106265.4 Da, respectively.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Table 1 and Supplementary Note (PDF 2741 kb)

Supplementary Data Set 1

DSS cross-links identified in the complex of S. elongatus RbcL8 and Raf1 (XLSX 76 kb)

Supplementary Data Set 2

Validation of Raf1 antibodies (PDF 197 kb)

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Hauser, T., Bhat, J., Miličić, G. et al. Structure and mechanism of the Rubisco-assembly chaperone Raf1. Nat Struct Mol Biol 22, 720–728 (2015). https://doi.org/10.1038/nsmb.3062

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