Complex Chaperone Dependence of Rubisco Biogenesis

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The Complex Chaperone Dependence of Rubisco Biogenesis Robert H Wilson, and Manajit Hayer-Hartl Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00132 • Publication Date (Web): 28 Mar 2018 Downloaded from http://pubs.acs.org on March 30, 2018

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Biochemistry

The Complex Chaperone Dependence of Rubisco Biogenesis

Robert H. Wilson and Manajit Hayer-Hartl*

Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany

*Correspondence: [email protected] (M.H-H.)

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ABSTRACT:

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), a ~550 kDa complex of eight large (RbcL) and eight small subunits (RbcS), mediates the fixation of atmospheric CO2 into usable sugars during photosynthesis. Despite its fundamental role, Rubisco is a remarkably inefficient enzyme and thus is produced by plants in huge amounts. It has long been a key target for bioengineering with the goal to increase crop yields. However, such efforts have been hampered by the complex requirement of Rubisco biogenesis for molecular chaperones. Recent studies have identified an array of auxiliary factors needed for the folding and assembly of the Rubisco subunits. The folding of plant RbcL subunits is mediated by the cylindrical chloroplast chaperonin, Cpn60, and its co-factor Cpn20. Folded RbcL requires a number of additional Rubisco specific assembly chaperones, including RbcX, Rubisco accumulation factors 1 (Raf1) and 2 (Raf2) and the Bundle sheath defective-2 (BSD2), to mediate the assembly of the RbcL8 intermediate complex. Incorporation of the RbcS and displacement of the assembly factors generates the active holoenzyme. An Escherichia coli strain expressing the chloroplast chaperonin and auxiliary factors now allows the expression of functional plant Rubisco, paving the way for Rubisco engineering by large scale mutagenesis. Here we review our current understanding on how these chaperones cooperate to produce one of the most important enzymes in nature.

INTRODUCTION:

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is an ancient enzyme found in cyanobacteria, algae and plants.1 It evolved more than 3.5 billion years ago in an atmosphere free of O2 and rich in CO2.2 Life on earth critically depends on the ability of photosynthetic organisms to convert inorganic carbon from CO2 into biomolecules. Rubisco catalyzes the carboxylation of 2 ACS Paragon Plus Environment

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Biochemistry

its five-carbon sugar substrate, ribulose-1,5-bisphosphate (RuBP), resulting – via an unstable sixcarbon intermediate – in two molecules of 3-phosphoglycerate, to be used for the synthesis of sugars, fatty acids and amino acids (Figure 1A). The carboxylation activity of Rubisco is slow (210 CO2 per second). As a consequence, plants may use up to 50% of their nitrogen to produce large amounts of the enzyme.3 Furthermore, Rubisco’s ability to distinguish between CO2 and the highly abundant O2 is limited, resulting in the wasteful process of photorespiration.4 Thus, Rubisco is a long-standing target for improving crop productivity.2, 5-8 Despite 50 years of effort, success in engineering plant Rubisco has been limited, due in part to the inability to recombinantly express the plant enzyme and perform large scale mutagenesis.

Figure 1. Rubisco: the key enzyme in photosynthesis. (A) The process of photosynthesis converts sunlight into chemical energy, splits water to liberate O2, and fixes CO2 for the synthesis of sugar, amino acids and fatty acids. The light reactions provide energy and reducing agents 3 ACS Paragon Plus Environment

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(ATP and NADPH), which are used in the light-independent Calvin-Benson-Bassham (CBB) cycle. The enzyme Rubisco catalyzes the key step of fixation of atmospheric CO2 by mediating the carboxylation of the 5-carbon sugar substrate ribulose-1,5-bisphosphate (RuBP). (B) Structure of hexadecameric form I Rubisco from spinach (PDB:1RCX).9 Side and top views shown in surface representation (RbcL subunits alternating in white and beige; RbcS subunits in magenta), with one antiparallel RbcL dimer (RbcL in green and RbcL’ in blue) and the adjacent RbcS subunits shown in ribbon representation. The antiparallel RbcL dimer is shown with RuBP (in red) bound in the active sites.

The major form of Rubisco (form I), including the plant enzyme, is a hexadecameric complex of ~ 530 kDa consisting of eight large (RbcL, ~50-55 kDa) and eight small (RbcS, ~1218 kDa) subunits.10, 11 The RbcL subunits form the core of the complex and are arranged as a tetramer of anti-parallel dimers, each dimer harbouring two catalytic sites (Figure 1B). The RbcL8 core cylinder is capped by four RbcS subunits at the top and four at the bottom. RbcL is always chloroplast encoded, but RbcS is nuclear encoded in plants and green algae. The simpler form II Rubisco in proteobacteria lacks RbcS and performs CO2 fixation as a dimer of RbcL subunits (RbcL2) or as multimer of dimers (RbcL4-Rbc10).12 It has long been known that the chloroplast chaperonin (Cpn60), a homolog of Escherichia coli (E. coli) GroEL and mitochondrial Hsp60, is involved in Rubisco biogenesis by interacting with RbcL.13, 14 In the late 1980’s it became clear that chaperonins mediate protein folding15, 16 and the form II Rubisco of the proteobacterium Rhodospirullum rubrum could be reconstituted from the denatured state using bacterial GroEL and its co-factor GroES.17 While the folded form II RbcL subunits assemble spontaneously to the functional dimer,18 form I Rubisco of cyanobacterial or plant origin could not be reconstituted in vitro using the E. coli chaperonins.19 The past ten years have seen the discovery and mechanistic analysis of several form I Rubisco specific auxiliary factors which are either essential for holoenzyme assembly or increase assembly efficiency.20 As a result Rubisco has become a paradigm for chaperoneassisted assembly. 4 ACS Paragon Plus Environment

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Biochemistry

Here, we review recent advances in understanding the chaperone machineries required for the biogenesis of plant Rubisco and its heterologous expression in a bacterial host. We will discuss the importance of understanding Rubisco biogenesis in the context of current initiatives towards engineering Rubisco.

Chaperonin-assisted RbcL folding

Chaperonins are ATP-regulated macromolecular machines that function as nano-compartments for single protein molecules to fold in isolation, unimpaired by aggregation.20, 21 GroEL and its homologues (chloroplast Cpn60 and mitochondrial Hsp60) form large cylindrical complexes consisting of two heptameric rings of ~60 kDa subunits that are stacked back to back. Whereas GroEL and Hsp60 are homo-oligomeric, Cpn60 is composed of homologous α and β subunits (Cpn60α7β7), with the β-subunits being able to form homo-oligomeric chaperonin complexes, while the α-subunits cannot assemble on their own.20, 22-24 These chaperonins functionally cooperate with single-ring co-factors that bind to the ends of the cylinder in a manner regulated by the chaperonin ATPase. These co-factors are heptameric rings of ~10 kDa: GroES in bacteria; Hsp10 in mitochondria and Cpn10 in chloroplasts. In addition, chloroplasts contain Cpn20, a tandem repeat of Cpn10 units that either function as a homo-tetramer or as a hetero-oligomer with Cpn10.25, 26


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Figure 2. The GroEL:GroES chaperonin system. (A) Left: Structure of the asymmetric GroELGroES-ADP7 complex (PDB:1AON).27 The three domains of each subunit: equatorial, intermediate and apical of one subunit each in the cis- and trans-ring of the tetradecameric GroEL are colored in magenta, yellow and blue, respectively, and one subunit of the heptameric GroES is colored in red. Adapted from ref. 28. Right: A cut-through of the GroEL-GroES-ADP7 complex showing polar and charged side-chain atoms in blue, hydrophobic side-chains atoms in yellow, backbone atoms in white and solvent-excluded surfaces of subunit interfaces in gray. Reproduced from ref. 27. (B) Model of the GroEL/GroES reaction cycle. Substrate protein (SP) binds to the open trans-ring of the GroEL-GroES-ADP7 complex via hydrophobic interactions with the apical domains. ATP binding to the SP-bound ring triggers conformational changes and this is followed by the binding of GroES and SP encapsulation for folding. Concomitant to ATPbinding the two rings of GroEL transiently separate, and ADP and GroES dissociation from the former cis-ring. Reassembly to the GroEL tetradecamer then occurs with or without exchange of 6 ACS Paragon Plus Environment

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Biochemistry

rings between GroEL complexes. SP remains encapsulated, free to fold, for the time needed to hydrolyze the 7 ATP molecules (2-7 s, dependent on temperature). Figure modified from ref. 29.

The mechanism of chaperonin-mediated folding has been studied extensively with the bacterial GroEL/GroES system.30 Briefly, unfolded substrate protein binds to the open ring of the GroEL/GroES-ADP complex via hydrophobic interactions with the apical domains of the GroEL subunits (Figure 2). Subsequent, ATP-dependent binding of GroES to the same ring displaces the bound protein in a cage formed by the GroEL-ring and the lid-like GroES. This step results in a conformational change that renders the cage hydrophilic. The encapsulated protein is now free to fold in isolation during the time required for hydrolysis (~2-7 s dependent on temperature). The folding cage opens upon ATP-binding to the opposite ring, allowing release of folded protein.30 Concomitantly, the two GroEL rings undergo transient separation with possible ring exchange between complexes (Figure 2B).29

Assembly Chaperones of Form I Rubisco

The failure of RbcL subunits of cyanobacterial form I Rubisco to assemble upon release from chaperonin in vitro suggested that one or more auxiliary factors may be required to assist the assembly process. A candidate for such a factor was the protein RbcX, which was identified due its conserved position within the cyanobacterial Rubisco operon of many species where it lies between, and is co-transcribed with, the rbcL and rbcS genes.31 Since loss of rbcX in some cyanobacterial species was observed to decrease Rubisco content, a role in Rubisco biogenesis was hypothesised.32-34 RbcX is a homodimer of ~15 kDa subunits and is conserved in all plants (Figure 3A).35-38 The hydrophobic groove in the center of the dimer binds the conserved ‘EIKFE(F/Y)D’ sequence motif within the RbcL C-terminal tail.35, 39 A crystal structure of the RbcX-bound assembly intermediate showed that RbcX clamps the RbcL2 unit and promotes 7 ACS Paragon Plus Environment

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assembly to a complex of RbcL8 with eight RbcX dimers bound (Figure 3A).40 Combining GroEL/ES chaperonin with RbcX allowed the first in vitro reconstitution of the cyanobacterial form I Rubisco.19 However, the reconstitution was of limited efficiency (~50% yield), suggesting the involvement of additional factors. Indeed, a screen of photosynthetic maize mutants identified several potential Rubisco assembly factors, including Rubisco accumulation factors 1 and 2 (Raf1and Raf2, respectively) and Bundle sheath defective-2 (BSD2).41 Like RbcS, RbcX and these other factors are nuclear encoded.


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Biochemistry

Figure 3. Auxiliary factors. (A) Left: Ribbon representation of the cyanobacterial RbcX dimer (PDB:2PEN).35 Two perpendicular views are shown. The two protomers are indicated in orange 9 ACS Paragon Plus Environment

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and yellow. Figure modified from ref. 20. Right: Crystal structure of a trapped RbcL8RbcX8 assembly intermediate (PDB:3RG6).40 The RbcL8 core structure is shown in surface representation and the bound RbcX in ribbon representation. The C-terminal tails of the RbcL subunits are bound within the central hydrophobic cleft of RbcX (indicated for one antiparallel RbcL dimer unit by red dashed circles). (B) Left: Crystal structures of the N-terminal α-helical domain (α-domain) (PDB:4WT3) and C-terminal β-sheet dimerization domain (β-domain) (PDB:4WT4) of Raf1 from A. thaliana.42 The protomers in the β-domain dimer are shown in blue and cyan. Right: 3D reconstruction of the RbcL8Raf14 complex (EMD-3053) from negative-stain electron microscopy images.42 Side view of the structural model is shown. Rigid-body domain fitting of a RbcL2 dimer and one Raf1 in the complex is superposed. The Raf1 dimer subunits (ribbon representation) are shown in blue and cyan, and the RbcL2 (schematic representation) in pale yellow. (C) Left: Crystal structure of bacterial α-carboxysome Rubisco accumulation factor 2, acRAF (PDB:4LOW).43 The protomers in the dimer are shown in red and salmon. Right: Sequence alignment of Raf2 proteins from A. thaliana (AtRaf2) and Thiomonas intermedia K12 (acRAF). 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 transit peptide for AtRaf2 is not shown. The Uniprot/genome accession codes for AtRaf2 and acRAF are Q9LU63 and D5X340, respectively. (D) Left: Ribbon representation of the A. thaliana BSD2 crystal structure (PDB:6EKB).44 Two perpendicular views are shown. The Zn centers and cysteine ligands are shown in space-filling and stick representation, respectively. Right: Crystal structure of the RbcL8:BSD28 complex (PDB:6EKC).44 BSD2 (green) is shown in ribbon representation. RbcL8 is shown in space filling with RbcL in white and RbcL’ in light orange. Raf1 is essential in plants41 and is also present in all cyanobacteria and algae that contain RbcX, possibly explaining why RbcX is not always essential in cyanobacteria.34 Remarkably, Raf1 alone proved to be highly efficient in mediating cyanobacterial Rubisco assembly, both in vitro and upon co-expression in E. coli.42, 45 Raf1, like RbcX, functions as a dimer. However, the Raf1 subunit is ~40 kDa in size and has no homology to RbcX. Each subunit consists of an Nterminal α-helical domain and a C-terminal β-sheet dimerization domain (Figure 3B).42 Similar to RbcX, Raf1 also stabilizes the RbcL2 unit with the β-sheet domain located at the equator of RbcL2 and the α-helical arms hugging the RbcL dimer at the top and bottom edges. The end-state of Raf1-mediated assembly is an RbcL8 core with four Raf1 dimers bound (Figure 3B).42 Raf2 shows distant homology to a class of enzymes called pterin-4a-carbinoloamine dehydratases (PCD), and shares the PCD domain with a potential homolog from alpha10 ACS Paragon Plus Environment

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Biochemistry

cyanobacteria, a ~9 kDa protein called acRAF (Figure 3C).43 The eukaryotic Raf2 homologs are considerably larger (~19 kDa), carrying a conserved N-terminal extension (Figure 3C) and also seem to function as homodimers like RbcX and Raf1.46 Evidence has been presented that Raf2 interacts with RbcS,46 but its exact role remains to be clarified. A recent study showed that in A. thaliana Raf2 is localized in the chloroplast stroma and A. thaliana mutant plants display reduced levels of Rubisco, in support of a critical role of Raf2 in Rubisco assembly.47 BSD2 was originally identified in a mutant maize line defective in bundle sheath development48 and was subsequently shown to be involved in Rubisco biogenesis and translational regulation.49-52 Unlike RbcX, Raf1 and Raf2, the ~8-10 kDa BSD2 is a chloroplastspecific zinc-finger protein. The crystal structure of Arabidopsis thaliana BSD2 was recently solved; it showed that BSD2 is an elongated monomer of hairpin architecture and two Zn atoms each coordinated by four cysteines (Figure 3D).44 Insight into the role of BSD2 in Rubisco assembly was provided by the crystal structure of a heterologous complex consisting of RbcL8 from the cyanobacterium Thermosynechococcus elongatus BP-1 and A. thaliana BSD2. This structure shows each RbcL2 unit being stabilized by two BSD2 molecules (Figure 3D).44 Binding to RbcL stabilises the otherwise unstructured C-terminus of BSD2, as these residues bind into the Rubisco active site and thus have the potential to prevent premature RuBP-binding. In summary, the structurally unrelated RbcX, Raf1 and BSD2 all function in promoting RbcL8 assembly by stabilizing the anti-parallel RbcL dimers, albeit by utilizing different binding regions on RbcL (Figure 3).

Insights from Recombinant Expression of Plant Rubisco

While the co-expression of RbcX or Raf1 allowed the functional recombinant expression of form I Rubisco from several cyanobacterial species,32-35, 45 efforts to express plant Rubisco had been 11 ACS Paragon Plus Environment

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unsuccessful. However, the functional expression of Rubisco from A. thaliana in E. coli was recently reported, demonstrating the requirement for co-expression of the cognate chloroplast chaperonin and auxiliary factors (Figure 4).44

Figure 4. Multiplicity of molecular chaperones involved in the biogenesis of plant Rubisco. Rubisco biogenesis requires coordination of cytosolic and chloroplast translation. The RbcL subunit is chloroplast-encoded and synthesized on ribosomes in the chloroplast stroma, while most chloroplast proteins in plants, including the folding and assembly factors, as well as RbcS, are nuclear-encoded and are synthesized on cytosolic ribosomes with a chloroplast targeting sequence (red). These proteins are imported into the chloroplast in an unfolded state.53 Following cleavage of the signal peptide by stromal signal peptidase, molecular chaperones in the stroma assist their folding to the functional state. In the stroma, folding of newly-synthesized RbcL subunits by the Cpn60αβ/Cpn20 chaperonin is followed by assembly to RbcL dimers and higher oligomers mediated by Raf1 and RbcX acting in cooperation or in parallel. Binding of BSD2 causes the displacement of these factors and stabilizes RbcL8 cores in a state competent for association with RbcS. RbcS binding causes displacement of BSD2, forming the functional holoenzyme. Raf2 is essential for Rubisco biogenesis and may act downstream or upstream of chaperonin. Figure modified from refs. 20 and 44.


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Biochemistry

The chaperonin system of E. coli (GroEL/ES) mediates the folding of ~10% of cytosolic proteins,54, 55 and is competent in folding the RbcL subunits of bacterial form II and cyanobacterial form I Rubisco. In light of the high degree of structural conservation of RbcL subunits between plants and cyanobacteria,10 it is surprising that GroEL/ES could not replace the chloroplast chaperonin system (Cpn60/Cpn20) for A. thaliana RbcL folding (AtRbcL).44 Coexpression of both Cpn60α and Cpn60β was required. We note that the Cpn60 subunits lack the C-terminal ‘GGM’ repeat motif present in GroEL and mitochondrial Hsp60, which is thought to interact with substrate proteins during binding and folding.56-58 In contrast to GroEL, the cofactor GroES efficiently replaced Cpn20, indicating that specificity for plant RbcL is conferred by the hetero-oligomeric Cpn60. Notably, the GroES-like Cpn10 failed to mediate AtRbcL folding and disrupted the function of Cpn20 when co-expressed. This latter effect may be attributed to formation of non-functional Cpn20/Cpn10 hetero-oligomers in the E. coli system.25 Why AtRbcL has a specific requirement for Cpn60 remains unclear, especially in light of chloroplast transformation experiments in tobacco, which have shown that Cpn60 is functionally compatible with bacterial RbcL.59 In addition to chaperonin, the recombinant production of plant Rubisco required the four auxiliary factors described above: Raf1, Raf2, RbcX, and BSD2 (Figure 4).44 Except for RbcX, which enhanced the yield, the other factors were essential under the conditions of E. coli expression, with individual omission of Raf1, Raf2 or BSD2 resulting in loss of holoenzyme production and RbcL aggregation. The end-state of RbcL assembly, in the absence of RbcS, was the RbcL8 complex with eight copies of BSD2 bound.44 Addition of RbcS to this complex resulted in the displacement of BSD2 and holoenzyme formation. Thus, BSD2 appears to have a special role in plant Rubisco assembly by maintaining RbcL8 in an inactive, assembly competent state when RbcS, which must be imported, is limiting (Figure 4). Consistent with previous 13 ACS Paragon Plus Environment

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findings that suggest BSD2 has a regulatory function in RbcL synthesis,49-52 it would seem plausible that accumulation of RbcL8:BSD28 has a role in this process. Interestingly, the formation of RbcL8:BSD28 (in the absence of RbcS) was critically dependent on Raf1 and Raf2 acting upstream in the assembly pathway. It must be concluded, therefore, that Raf2 also acts on RbcL,44 although an additional interaction with RbcS is not ruled out.43, 46 Co-expression of RbcL and RbcS from tobacco (Nicotiana tabacum) with A. thaliana chaperonin and auxiliary factors failed to produce any significant amount of the tobacco Rubisco,44 although AtRbcL and NtRbcL share ~94% sequence identity (96% similarity). The amount of tobacco Rubisco increased, reaching ~10% of the yield of the A. thaliana enzyme, when the AtRaf1 was replaced by NtRaf1 (~55% identity/70% similarity). This is consistent with the finding that expression of A. thaliana Rubisco in tobacco chloroplast was augmented by AtRaf1 co-expression,60 and suggests that the auxiliary factors have co-evolved with their cognate Rubisco.

Conclusion and Outlook

Recent advances in understanding the complexity of form I Rubisco biogenesis has cumulated in the ability to recombinantly express the plant enzyme in a bacterial host. Although it seems unlikely that major Rubisco assembly factors have remained undiscovered, the exact assembly pathway remains to be defined. Reconstitution of plant Rubisco in vitro with all purified components is an important goal of future research and will help to define the contribution of each of the assembly factors in the overall reaction.61 Such experiments will also reveal the promiscuity range for chaperone-client relationships. Determining the stringency of compatibility for each factor will provide information about the evolutionary constrains of the enzyme and allow researchers to carefully curate heterologous expression systems for Rubisco engineering. 14 ACS Paragon Plus Environment

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Biochemistry

Can plant Rubisco now be engineered to improve the enzyme for agricultural use? Several platforms for Rubisco engineering by directed evolution62 have been developed and may exploit the recent advance in recombinantly expressing the plant enzyme. For this purpose the plant Rubisco biogenesis machinery can be incorporated into Rubisco-dependent E. coli (RDE) screens to identify mutants with improved functional properties.8, 63 Previous work with cyanobacterial Rubisco in RDE showed that co-expression of assembly chaperones such as RbcX may impede the evolutionary potential of RbcL.64 The extensive chaperone requirement of plant Rubisco expression may further reduce the sequence space that can be explored to make a functional enzyme, providing a possible explanation for why Rubisco has been unable to improve its functional shortcomings through natural evolution.65-68 However, modulating the expression of chaperones in RDE, or subjecting the chaperones themselves to a mutational screen has the exciting potential to take Rubisco across unexplored evolutionary trajectories.

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] ORCID

Manajit Hayer-Hartl: 0000-0001-8213-6742 Author Contributions

R.H.W. and M.H.-H. contributed equally to this work. Funding

M.H.-H. acknowledges funding by the Minerva Foundation of the Max Planck Society and the Deutsche Forschungsgemeinschaft (SFB 1035). Notes

The authors declare no competing financial interest.

ACKNOWLEDGEMENTS


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The authors thank A. Bracher for providing us the scripts for producing the structural figures and F. Ulrich Hartl for critically reading the manuscript.

REFERENCES

(1) Nisbet, E. G., Grassineau, N. V., Howe, C. J., Abell, P. I., Regelous, M., and Nisbet, R. E. R. (2007) The age of Rubisco: the evolution of oxygenic photosynthesis. Geobiology 5, 311-335. (2) Whitney, S. M., Houtz, R. L., and Alonso, H. (2011) Advancing our understanding and capacity to engineer nature's CO2-sequestering enzyme, Rubisco. Plant Physiol. 155, 27-35. (3) Spreitzer, R. J., and Salvucci, M. E. (2002) Rubisco: structure, regulatory interactions, and possibilities for a better enzyme. Annu. Rev. Plant Biol. 53, 449-475. (4) Maurino, V. G., and Peterhansel, C. (2010) Photorespiration: current status and approaches for metabolic engineering. Curr. Opin. Plant Biol. 13, 249-256. (5) Furbank, R. T., Quick, W. P., and Sirault, X. R. R. (2015) Improving photosynthesis and yield potential in cereal crops by targeted genetic manipulation: Prospects, progress and challenges. Field Crops Res. 182, 19-29. (6) Erb, T. J., and Zarzycki, J. (2016) Biochemical and synthetic biology approaches to improve photosynthetic CO2-fixation, Curr. Opin. Chem. Biol. 34, 72-79. (7) Orr, D., Alcântara, A., Kapralov, M. V., Andralojc, J., Carmo-Silva, E., and Parry, M. A. J. (2016) Surveying Rubisco diversity and temperature response to improve crop photosynthetic efficiency. Plant Physiol. 172, 707-717. (8) Conlan, B., and Whitney, S. (2018) Preparing Rubisco for a tune up. Nat. Plants 4, 12-13. (9) Taylor, T. C., and Andersson, I. (1997) The structure of the complex between rubisco and its natural substrate ribulose 1,5-bisphosphate. J. Mol. Biol. 265, 432-444. (10) Andersson, I., and Backlund, A. (2008) Structure and function of Rubisco. Plant Physiol. Biochem. 46, 275-291. (11) Tabita, F. R., Satagopan, S., Hanson, T. E., Kreel, N. E., and Scott, S. S. (2008) Distinct form I, II, III, and IV Rubisco proteins from the three kingdoms of life provide clues about Rubisco evolution and structure/function relationships. J. Exp. Bot. 59, 1515-1524. (12) Tabita, F. R., Hanson, T. E., Li, H., Satagopan, S., Singh, J., and Chan, S. (2007) Function, structure, and evolution of the RubisCO-like proteins and their RubisCO homologs. Microbiol. Mol. Biol. Rev. 71, 576-599.


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Biochemistry

(13) Barraclough, R., and Ellis, R. J. (1980) Protein synthesis in chloroplasts. IX. Assembly of newly-synthesized large subunits into ribulose bisphosphate carboxylase in isolated intact pea chloroplasts. Biochim. Biophys. Acta 608, 18-31. (14) Roy, H., Bloom, M., Milos, P., and Monroe, M. (1982) Studies on the assembly of large subunits of ribulose bisphosphate carboxylase in isolated pea chloroplasts. J. Cell Biol. 94, 20-27. (15) Ostermann, J., Horwich, A. L., Neupert, W., and Hartl, F.-U. (1989) Protein folding in mitochondria requires complex formation with Hsp60 and ATP hydrolysis. Nature 341, 125-130. (16) Martin, J., Langer, T., Boteva, R., Schramel, A., Horwich, A. L., and Hartl, F. U. (1991) Chaperonin-mediated protein folding at the surface of GroEL through a 'molten globule'-like intermediate. Nature 352, 36-42. (17) Goloubinoff, P., Gatenby, A. A., and Lorimer, G. H. (1989) GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli. Nature 337, 44-47. (18) Brinker, A., Pfeifer, G., Kerner, M. J., Naylor, D. J., Hartl, F. U., and Hayer-Hartl, M. (2001) Dual function of protein confinement in chaperonin-assisted protein folding. Cell 107, 223-233. (19) Liu, C., Young, A. L., Starling-Windhof, A., Bracher, A., Saschenbrecker, S., Rao, B. V., Rao, K. V., Berninghausen, O., Mielke, T., and Hartl, F. U. (2010) Coupled chaperone action in folding and assembly of hexadecameric Rubisco. Nature 463, 197-202. (20) Bracher, A., Whitney, S. M., Hartl, F. U., and Hayer-Hartl, M. (2017) Biogenesis and metabolic maintenance of Rubisco. Annu. Rev. Plant Biol. 68, 29-60. (21) Balchin, D., Hayer-Hartl, M., and Hartl, F. U. (2016) In vivo aspects of protein folding and quality control. Science 353, aac4354. (22) Vitlin Gruber, A., Nisemblat, S., Azem, A., and Weiss, C. (2013) The complexity of chloroplast chaperonins. Trends Plant Sci. 18, 688-694. (23) Zhang, S., Zhou, H., Yu, F., Bai, C., Zhao, Q., He, J., and Liu, C. (2016) Structural insight into the cooperation of chloroplast chaperonin subunits. BMC Biol. 14, 29. (24) Zhao, Q., and Liu, C. (2018) Chloroplast chaperonin: An intricate protein folding machine for photosynthesis. Front. Mol. Biosci. 4, 98. (25) Tsai, Y.-C. C., Mueller-Cajar, O., Saschenbrecker, S., Hartl, F. U., and Hayer-Hartl, M. (2012) Chaperonin cofactors, Cpn10 and Cpn20, of green algae and plants function as heterooligomeric ring complexes. J. Biol. Chem. 287, 20471-20481. (26) Vitlin Gruber, A., Zizelski, G., Azem, A., and Weiss, C. (2014) The Cpn10(1) co-chaperonin of A. thaliana functions only as a hetero-oligomer with Cpn20. PLoS One 9, e113835. 17 ACS Paragon Plus Environment

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(27) Xu, Z., Horwich, A. L., and Sigler, P. B. (1997) The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex. Nature 388, 741-750. (28) Hartl, F. U., and Hayer-Hartl, M. (2002) Molecular chaperones in the cytosol: from nascent chain to folded protein, Science 295, 1852-1858. (29) Yan, X., Shi, Q., Bracher, A., Milicic, G., Singh, A. K., Hartl, F. U., and Hayer-Hartl, M. (2018) GroEL ring separation and exchange in the chaperonin reaction. Cell 172, 605-617. (30) Hayer-Hartl, M., Bracher, A., and Hartl, F. U. (2016) The GroEL-GroES chaperonin machine: A nano-cage for protein folding. Trends Biochem. Sci. 41, 62-76. (31) Larimer, F. W., and Soper, T. S. (1993) Overproduction of Anabaena 7120 ribulosebisphosphate carboxylase/oxygenase in Escherichia coli. Gene 126, 85-92. (32) Li, L.-A., and Tabita, F. R. (1997) Maximum activity of recombinant ribulose 1, 5bisphosphate carboxylase/oxygenase of Anabaena sp. strain CA requires the product of the rbcX gene. J. Bacteriol. 179, 3793-3796. (33) Onizuka, T., Endo, S., Akiyama, H., Kanai, S., Hirano, M., Yokota, A., Tanaka, S., and Miyasaka, H. (2004) 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. (34) Emlyn-Jones, D., Woodger, F. J., Price, G. D., and Whitney, S. M. (2006) RbcX can function as a Rubisco chaperonin, but is non-essential in Synechococcus PCC7942. Plant Cell Physiol. 47, 1630-1640. (35) Saschenbrecker, S., Bracher, A., Rao, K. V., Rao, B. V., Hartl, F. U., and Hayer-Hartl, M. (2007) Structure and function of RbcX, an assembly chaperone for hexadecameric Rubisco. Cell 129, 1189-1200. (36) Kolesinski, P., Golik, P., Grudnik, P., Piechota, J., Markiewicz, M., Tarnawski, M., Dubin, G., and Szczepaniak, A. (2013) Insights into eukaryotic Rubisco assembly - crystal structures of RbcX chaperones from Arabidopsis thaliana. Biochim. Biophys. Acta 1830, 2899-2906. (37) Tarnawski, M., Krzywda, S., Bialek, W., Jaskolski, M., and Szczepaniak, A. (2011) Structure of the RuBisCO chaperone RbcX from the thermophilic cyanobacterium Thermosynechococcus elongatus. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 67, 851857. (38) Bracher, A., Hauser, T., Liu, C. M., Hartl, F. U., and Hayer-Hartl, M. (2015) Structural analysis of the Rubisco-assembly chaperone RbcX-II from Chlamydomonas reinhardtii. PLoS One 10, e0135448 (39) Tabita, F. R. (2007) Rubisco: The enzyme that keeps on giving. Cell 129, 1039-1040. 18 ACS Paragon Plus Environment

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(51) Salesse-Smith, C., Sharwood, R. E., Sakamoto, W., and Stern, D. B. (2017) The Rubisco chaperone BSD2 may regulate chloroplast coverage in maize bundle sheath cells. Plant Physiol. 175, 1624-1633. (52) Doron, L., Segal, N. a., Gibori, H., and Shapira, M. (2014) The BSD2 ortholog in Chlamydomonas reinhardtii is a polysome‐associated chaperone that co‐migrates on sucrose gradients with the rbcL transcript encoding the Rubisco large subunit. Plant J. 80, 345-355. (53) Jarvis, P., and Kessler, F. (2014) Mechanisms of chloroplast protein import in plants. In: Advances in Plant Biology: Plastid Biology (S.M. Theg and F.-A. Wollman, eds.), Springer, New York, pp. 241-270. (54) Kerner, M. J., Naylor, D. J., Ishihama, Y., Maier, T., Chang, H. C., Stines, A. P., Georgopoulos, C., Frishman, D., Hayer-Hartl, M., Mann, M., and Hartl, F. U. (2005) Proteomewide analysis of chaperonin-dependent protein folding in Escherichia coli. Cell 122, 209-220. (55) Ewalt, K. L., Hendrick, J. P., Houry, W. A., and Hartl, F. U. (1997) In vivo observation of polypeptide flux through the bacterial chaperonin system. Cell 90, 491-500. (56) Tang, Y.-C., Chang, H.-C., Roeben, A., Wischnewski, D., Wischnewski, N., Kerner, M. J., Hartl, F. U., and Hayer-Hartl, M. (2006) Structural features of the GroEL-GroES nano-cage required for rapid folding of encapsulated protein. Cell 125, 903-914. (57) Chen, D.-H., Madan, D., Weaver, J., Lin, Z., Schroder, G. F., Chiu, W., and Rye, H. S. (2013) Visualizing GroEL/ES in the act of encapsulating a folding protein. Cell 153, 1354-1365. (58) Weaver, J., and Rye, H. S. (2014) The C-terminal tails of the bacterial chaperonin GroEL stimulate protein folding by directly altering the conformation of a substrate protein. J. Biol. Chem. 289, 23219-23232. (59) Whitney, S. M., and Sharwood, R. E. (2008) Construction of a tobacco master line to improve Rubisco engineering in chloroplasts. J. Exp. Bot. 59, 1909-1921. (60) Whitney, S. M., Birch, R., Kelso, C., Beck, J. L., and Kapralov, M. V. (2015) Improving recombinant Rubisco biogenesis, plant photosynthesis and growth by coexpressing its ancillary RAF1 chaperone. Proc. Natl. Acad. Sci. U. S. A. 112, 3564-3569. (61) Wilson, R., and Whitney, S. (2015) Photosynthesis: Getting it together for CO2 fixation, Nat. Plants 1, 15130. (62) Wilson, R. H., and Whitney, S. M. (2017) Improving CO2 fixation by enhancing Rubisco performance. In Directed Enzyme Evolution: Advances and Applications (M. Alcalde, ed.), Springer, New York, pp 101-126. (63) Wilson, R. H., Martin-Avila, E., Conlan, C., and Whitney, S. M. (2018) An improved Escherichia coli screen for Rubisco identifies a protein-protein interface that can enhance CO2fixation kinetics. J. Biol. Chem. 293, 18-27. 20 ACS Paragon Plus Environment

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Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For Table of Contents Use Only

The Complex Chaperone Dependence of Rubisco Biogenesis Robert H. Wilson and Manajit Hayer-Hartl*


Page 22 of 27

Page 23 of 27

Biochemistry

Wilson & Hayer-Hartl, Figure 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

B

Sunlight

RbcS

side-view

O2

H2O

RbcL’

RbcL RuBP

Light reactions 90o NADPH Chemical NADP ATP energy ADP RbcL

CO2

CBB cycle Rubisco

top-view

RuBP Sugar Amino acids Fatty acids

ACS Paragon Plus Environment

RbcS

Biochemistry

Page 24 of 27

Wilson & Hayer-Hartl, Figure 2 A GroES

cis-ring (hydrophilic)

GroEL

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

trans-ring (hydrophobic)

B

Transient ring separation and reassembly ATP trans

GroEL

ATP ADP

2-7 s

ATP

ADP

ATP

cis ADP

GroES

SP:

180o Unfolded Intermediate Folded


ATP

ADP

ADP

Page 25 of 27

Biochemistry

Wilson & Hayer-Hartl, Figure 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

RbcX (dimer; ~15 kDa a subunits) C

RbcL8:RbcX8

3 3

2

2

N

1

1 N

90º

4

4 C

C

B

Raf1 (dimer; ~40 kDa subunits)

N

RbcL8:Raf14

C

N Raf1 (dimerization domain) C Raf1 domain

C

Raf2 (dimer; ~9-19 kDa subunits)

Sequence alignment AtRaf2 and acRaf 1

C

10

20

30

40

50

60

AtRaf2 M D F L G D F G A R D P Y P E E I A S Q F G D K V L G C Q S T E H K I L I P N A S V L S L S Q L Q C S P V S S S Q P P L acRAF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . consensus>70 70

80

90

100

110

AtRaf2 S G D D A R T L L H K V L G W S I V D N E A G G L K I R C M W K V R D F G C . . . G V E L I N R I H K V A E A S G H Y P acRAF . . . . . . . . . . . . M S W R E Q G K P . . . . . . P M L F K R F A F G S Y A Q T R A F L D A L A A L S E E T G Q H P consensus>70

N

N

D

120

130

140

150

160

AtRaf2 S L H L E S P T Q V R A E L F T S S I G G L S M N D F I M A A K I D D I K T S D L S P R K R A W A . . . . . . . . . . . acRAF Q N I N F G T T . . . . . . . . . . . . . . . . . . . Y V N I T L D A A D G A T L G E A E R A F A A R V D A L A G S S G consensus>70

RbcL8:BSD28

BSD2 (monomer; ~8 kDa)

Zn2 1

2 90º

Zn1

N

N

C C


Biochemistry

Page 26 of 27

Wilson & Hayer-Hartl, Figure 4 1 2 3 4 Nuclear 5 genome 6 7 8 9 10 11 12 13 14 15 16 Chaperonin 17 Raf2? Cpn60 18 Ribosome 19 rbcL 20 mRNA 21 22 23 24 Nascent chain 25 Cpn20 26 binding chaperones? RbcL 27 28 29 30 TRANSLATION FOLDING 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Cytosol

Ribosome

mRNA bsd2 cpn60 rbcX cpn60 raf1 cpn20 raf2 cpn10 rbcS other proteins

Cytosolic nascent chain binding chaperones

Chloroplast stroma

Signal peptide

Chaperone-assisted folding of RbcS and auxiliary factors Raf1 RbcL2Raf1

BSD2

RbcS

Raf2? RbcX

BSD2

Raf1/RbcX X /Raf2 RbcL8BSD28 RbcL2RbcX2 ASSEMBLY


RbcL8RbcS8

Page 27 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

&KDSHURQLQ &SQ DE

5LERVRPH

GLIIHUHQW FKDSHURQHV 5EF; 5DI 5DI %6'

5XELVFR VPDOO VXEXQLW 5EF6

P51$

5XELVFR ASSEMBLY 5EF/ 5EF6

&SQ 5XELVFR ODUJH VXEXQLW 5EF/

TRANSLATION

FOLDING


Complex Chaperone Dependence of Rubisco Biogenesis

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