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Coupled chaperone action in folding and assembly of hexadecameric Rubisco

Abstract

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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Acknowledgements

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.

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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). https://doi.org/10.1038/nature08651

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